Pharmaceuticals for the treatment or prevention of macrophage-mediated diseases and their applications

Abstract

The present invention relates to a pharmaceutical product for treating or preventing macrophage-mediated diseases and its application. Specifically, the present invention provides the use of amniotic fluid in the manufacture of a pharmaceutical product for treating and / or preventing macrophage-mediated diseases. The amniotic fluid is derived from eggs of embryonic day 5 to 12, or from eggs of birds other than chickens whose developmental stage corresponds to that of eggs of the aforementioned embryonic ages, or from rodent embryos of embryonic day 8 to 14, or from embryos of mammals other than rodents whose developmental stage corresponds to that of rodents of embryonic day 8 to 14.

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical agent for the treatment or prevention of macrophage-mediated diseases and its application. [Background technology]

[0002] Macrophages express different types of macrophages in response to various stimuli, inducibly expressing higher levels of inducible nitric oxide synthase (iNOS) or arginase, and are accordingly called M1 and M2 macrophages. In inflammatory diseases, M1 macrophages accumulate at early inflammatory sites and are activated by inflammatory cytokines such as LPS, TNFα, and IFNγ. They then secrete inflammatory factors such as IL-12, promoting the onset and progression of inflammation and protecting the body from foreign invaders. In the later stages of inflammation, M2 cells play roles in inflammation suppression, tissue repair, and tissue structural reconstruction. The ratio of M1 / M2 macrophage populations during the onset and progression of inflammation continuously changes over time, eventually leading to complete resolution of the effects of inflammation. However, during some chronic inflammation and certain acute inflammatory processes, an imbalance in the ratio of M1 / M2 cell populations can occur, and excessive activation of the M1 cell population can cause significant tissue damage and subsequent adverse symptoms, such as a more severe cytokine storm. Therefore, M2 macrophages are closely related to anti-inflammatory responses and immune homeostasis, and are involved in tissue repair, tissue and organ reconstruction, scar formation, and wound healing, and exhibit immunosuppressive effects.

[0003] Various exosome-derived miRNAs, transcription factors, and other non-coding RNAs regulate M2 macrophage polarization through different signaling pathways, among which the Toll-like receptor 4 (TLR4) / NF-κB signaling pathway plays a central regulatory role in inflammatory responses. In the inactive state, NF-κB activity is inhibited by IκB. However, upon stimulation by cytokine receptors such as TLRs and TNF receptors, IκB is subsequently phosphorylated by the activation of IκB kinase, which then degrades the proteasome, releasing NF-κB, which then translocates to the nucleus and activates the transcription of corresponding genes. NF-κB activation can induce the synthesis and release of proinflammatory cytokines.

[0004] With the increasing incidence of diabetes, diabetic ulcers have become one of the most common chronic, intractable wounds clinically, and persistent chronic inflammation is a typical feature of diabetic skin wounds. Because the microenvironment of diabetic wounds is complex, including hypoxia, infection, ischemia, inflammation, and oxidative stress, diabetic ulcers tend to prolong and recur, and conventional debridement and dressing change therapy has limited effectiveness in treating diabetic wounds. Therefore, how to effectively and economically treat diabetic ulcers is an urgent issue that needs to be addressed.

[0005] During wound healing, M1 macrophages are responsible for phagocytosing necrotic tissue and cellular debris, while M2 macrophages are involved in suppressing inflammation and promoting tissue regeneration. Diabetic wounds are typically in a state of excessive inflammation, with a large number of M1 macrophages present in the wound tissue, and macrophage transformation to the M2 type is inhibited. It should be noted that previous studies have shown that an increase in the M2 phenotype may be an important factor in diabetic wound repair. Summary of the Invention

[0006] A first aspect of the present invention provides the use of amniotic fluid in the manufacture of a medicament for treating and / or preventing a macrophage-mediated disease, wherein the amniotic fluid is derived from an egg of embryonic day 5 to 12, preferably an egg of embryonic day 6 to 11, more preferably an egg of embryonic day 7 to 9, even more preferably an egg of embryonic day 7 to 8, or an egg of a bird other than chicken whose developmental period corresponds to that of the eggs of the above embryonic ages, or an embryo of a rodent of embryonic day 8 to 14, or an embryo of a mammal other than a rodent, other than a human, whose developmental period corresponds to that of an embryonic day 8 to 14.

[0007] The present invention also provides the use of amniotic fluid in the manufacture of a medicament for treating and / or preventing an M1 macrophage-mediated disease, wherein the amniotic fluid is derived from an egg of embryonic day 5 to 12, preferably an egg of embryonic day 6 to 11, more preferably an egg of embryonic day 7 to 9, even more preferably an egg of embryonic day 7 to 8, or an egg of a bird other than chicken whose developmental stage corresponds to that of the eggs of the above embryonic ages, or an embryo of a rodent of embryonic day 8 to 14, or an embryo of a mammal other than a human other than a rodent whose developmental stage corresponds to that of an embryonic day 8 to 14.

[0008] The present invention also provides use of amniotic fluid in the manufacture of a medicament for treating and / or preventing an M2 macrophage-mediated disease, wherein the amniotic fluid is derived from an egg of embryonic day 5 to 12, preferably an egg of embryonic day 6 to 11, more preferably an egg of embryonic day 7 to 9, even more preferably an egg of embryonic day 7 to 8, or an egg of a bird other than chicken whose developmental stage corresponds to that of the eggs of the above embryonic ages, or an embryo of a rodent of embryonic day 8 to 14, or an embryo of a mammal other than a human other than a rodent whose developmental stage corresponds to that of an embryonic day 8 to 14.

[0009] The present invention also provides use of amniotic fluid in the manufacture of a preparation for treating and / or preventing a disease mediated by the TLR4 / NF-κB signaling pathway, wherein the amniotic fluid is derived from an egg of embryonic day 5 to 12, preferably an egg of embryonic day 6 to 11, more preferably an egg of embryonic day 7 to 9, even more preferably an egg of embryonic day 7 to 8, or an egg of a bird other than chicken whose developmental period corresponds to that of the eggs of the above embryonic ages, or an embryo of a rodent of embryonic day 8 to 14, or an embryo of a mammal other than a human other than a rodent whose developmental period corresponds to that of a rodent of embryonic day 8 to 14.

[0010] The present invention also provides the use of amniotic fluid in the manufacture of a preparation for suppressing the number of M1 macrophage populations and increasing the proportion of M2 macrophages, or for promoting the transformation of M1 macrophages into M2 macrophages, wherein the amniotic fluid is derived from eggs of embryonic day 5 to 12, preferably from eggs of embryonic day 6 to 11, more preferably from eggs of embryonic day 7 to 9, and even more preferably from eggs of embryonic day 7 to 8, or from eggs of birds other than chickens whose developmental period corresponds to that of eggs of the above embryonic ages, or from rodent embryos of embryonic day 8 to 14, or from embryos of mammals other than rodents whose developmental period corresponds to that of rodents of embryonic day 8 to 14.

[0011] In one or more embodiments, the medicament or preparation is a cell culture comprising the amniotic fluid and / or embryonic stem cells.

[0012] In one or more embodiments, the medicament or preparation is a pharmaceutical composition comprising the amniotic fluid and / or chicken embryonic stem cells and a pharmaceutically acceptable excipient.

[0013] In one or more embodiments, the macrophage-mediated disease is selected from the group consisting of hypertrophic scars, chronic obstructive pulmonary disease, tumors such as breast cancer and liver cancer, metabolic diseases such as severe obesity, insulin resistance and type 2 diabetes, inflammatory diseases such as acute pancreatitis and arteriosclerosis, cardiovascular diseases such as myocarditis, myocardial infarction and arrhythmia, neurological disorders such as Alzheimer's disease, brain diseases such as cerebral infarction and brain injury, and eye diseases such as autoimmune uveitis, retinopathy, keratitis, corneal transplantation, Sjogren's syndrome, uveal melanoma and myopia. inflammatory bowel disease, autoimmune hepatitis, asthma, alcoholic liver disease, colitis, multiple sclerosis, periodontitis and other immune inflammatory diseases; arthritis such as rheumatoid arthritis, bone erosion, synovitis, osteoarthritis and other arthritis; acute kidney injury, chronic kidney disease, end-stage renal failure, proliferative glomerulonephritis, membranous nephropathy, diabetic nephropathy, purpura nephritis, ANCA-associated small vasculitis, urinary tract infection, nephritis such as autosomal dominant polycystic kidney disease; bacterial infections such as sepsis; pregnancy-induced hypertension, diabetes, gestational diabetes, diabetic nephropathy.

[0014] In one or more embodiments, the M1 macrophage-mediated disease is selected from tumors such as involuting hypertrophic scar, chronic obstructive pulmonary disease, breast cancer, and liver cancer; metabolic diseases such as severe obesity, insulin resistance, and type 2 diabetes; inflammatory diseases such as acute pancreatitis; coronary artery disease such as arteriosclerosis; kidney disease, cardiovascular diseases such as obesity, myocarditis, and myocardial infarction; brain diseases such as cerebral infarction and brain injury; eye diseases such as autoimmune uveitis, retinopathy, keratitis, corneal transplant, Sjögren's syndrome, and uveal melanoma; immune-inflammatory diseases such as inflammatory bowel disease, autoimmune hepatitis, asthma, alcoholic liver disease, colitis, multiple sclerosis, periodontitis, and osteoarthritis; diabetes, gestational diabetes, and diabetic nephropathy.

[0015] In one or more embodiments, the M2 macrophage-mediated disease is selected from tumors such as proliferative hypertrophic scar, breast cancer, and liver cancer; metabolic diseases such as insulin resistance and type 2 diabetes; inflammatory diseases such as acute pancreatitis; cardiovascular diseases such as myocardial infarction, myocardial failure, arteriosclerosis, coronary artery disease, and myocarditis; ophthalmological diseases such as cerebral infarction, autoimmune uveitis, retinopathy, keratitis, corneal transplant, Sjogren's syndrome, and uveal melanoma; immunoinflammatory diseases such as inflammatory bowel disease, autoimmune hepatitis, asthma, alcoholic liver disease, colitis, multiple sclerosis, periodontitis, and osteoarthritis; diabetes, gestational diabetes, and diabetic nephropathy.

[0016] In one or more embodiments, the TLR4 / NF-κB signaling pathway-mediated disease is selected from the group consisting of vascular inflammation such as systemic lupus erythematosus, arteriosclerosis, and coronary artery disease, myocarditis such as myocardial ischemic tissue inflammation and myocardial injury, hepatitis such as liver failure, alcoholic liver injury, and inflammatory immune response during alcohol metabolism, fatty liver, pneumonia such as acute lung injury, chronic obstructive pulmonary disease, and silicosis, nephritis such as acute kidney injury and lupus nephritis, inflammatory bowel disease such as acute enteritis, ulcerative colitis, and radiation proctitis, gastritis such as chronic atrophic gastritis, pneumonia, and gastrointestinal disease. The drug is selected from acute respiratory infections such as bronchitis, pharyngitis, sinusitis, and otitis media; periodontitis, hyperuricemia, allergic rhinitis, hypersensitivity rhinitis, mastitis, arthritis such as acute gouty arthritis, chronic arthritis, and rheumatoid arthritis; inflammation of wound tissue, hypertrophic scar, polycystic ovary syndrome, tumors such as pituitary prolactin adenoma, adrenocorticotropic hormone adenoma, and intracranial aneurysm; infectious diseases such as bacterial infection, fungal infection, and viral infection; and allergic diseases such as allergic skin diseases, bronchial asthma, hypersensitivity rhinitis, and allergic purpura. [Brief explanation of the drawings]

[0017] [Figure 1] Figure 1 is a schematic diagram showing that ceAF reduces inflammation in lipopolysaccharide-stimulated Raw264.7 cells via the TLR4 / NF-κB signaling pathway. In D, each group consists of four bars, and the bars in each group correspond to CD206, Arg-1, iNOS, TNF-α, IL-6, IL-1β, TLR4, NF-κB, and pIκB, respectively, from left to right. [Figure 2] FIG. 2 is a schematic diagram showing that ceAF induces polarization of RAW264.7 into M2 macrophages in vitro. [Figure 3] FIG. 3 is a schematic diagram showing that ceAF promotes wound healing in STZ-induced diabetic mice. [Figure 4] FIG. 4 is a schematic diagram showing that ceAF improves wound histological parameters in STZ-induced diabetic mice. [Figure 5] FIG. 5 is a schematic diagram showing that ceAF improves wound healing-related indicators in STZ-induced diabetic mice. [Figure 6] Figure 6 is a schematic diagram showing that ceAF can regulate wound-associated inflammatory factors in STZ-induced diabetic mice. In B, each group consists of two bars, and the bars in each group correspond to CD206, Arg-1, iNOS, TNF-α, IL-6, and IL-1β, from left to right. DETAILED DESCRIPTION OF THE INVENTION

[0018] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (e.g., in the Examples) can be combined with each other to form preferred means for solving the problems.

[0019] The present inventors discovered that chicken embryo amniotic fluid (ceAF) can induce the transformation of macrophages from M1 to M2 type via the TLR4 / NF-κB signaling pathway, and thus completed the present invention.

[0020] amniotic fluid Amniotic fluid can be obtained from ascent eggs and mammals other than humans. Ascent eggs refer to avian eggs. Preferred avian species include poultry such as chickens, ducks, and geese. In the present invention, poultry eggs with an embryonic age of 5 to 20 days, more preferably 6 to 15 days, are used. It should be understood that the appropriate embryonic age may vary depending on the poultry egg. For example, when chicken eggs are used, chicken eggs with an embryonic age of 5 to 12 days, more preferably 6 to 11 days, more preferably 7 to 9 days, and even more preferably 7 to 8 days are used. When eggs of other poultry species are used, eggs with a developmental stage corresponding to that of chicken eggs of the above embryonic ages may be used. For example, when duck eggs are used, duck eggs with an embryonic age of 8 to 10 days, particularly 8 to 9 days, are considered optimal. In some specific embodiments, ceAF refers to amniotic fluid extracted from chicken embryos with an embryonic age of 6 to 8 days.

[0021] Amniotic fluid from poultry eggs can be obtained by conventional methods. For example, the blunt end of an egg of the appropriate embryonic age is struck to break the eggshell, peeling it off and creating a hole approximately 2 cm in diameter. Next, the shell membrane and vitelline membrane are carefully peeled off with tweezers, taking care not to damage the amniotic membrane. The amniotic membrane and its connective tissue surrounding the embryo are poured from the shell into a petri dish, and the amniotic membrane is punctured with a syringe to extract amniotic fluid until the amniotic membrane adheres tightly to the embryo, thereby obtaining the amniotic fluid used in the present invention.

[0022] As used herein, amniotic fluid may be derived from non-human mammals, particularly rodents such as mice. Other non-human mammals may include common livestock such as cows, sheep, dogs, cats, and pigs. In certain embodiments, amniotic fluid is derived from a rodent embryo at 8-14 days of gestation, or from a non-human mammal embryo whose developmental stage corresponds to that of a rodent at 8-14 days of gestation. Amniotic fluid can be obtained by conventional methods. For example, the abdominal cavity of a mouse at 8-14 days of gestation is opened with surgical scissors, the uterus is carefully removed and incised, and the amniotic membrane is punctured with a syringe to extract amniotic fluid until it adheres tightly to the embryo. This allows the amniotic fluid used in the present invention to be obtained.

[0023] It should be understood that, if necessary, the amniotic fluid is centrifuged to separate possible impurities such as egg yolk and obtain amniotic fluid as pure as possible. The supernatant obtained after centrifugation is the amniotic fluid used in the present invention. All steps for obtaining amniotic fluid must be performed under sterile conditions, and it should be understood that the term "amniotic fluid" used herein refers to "pure" amniotic fluid, i.e., amniotic fluid isolated from poultry eggs or non-human mammalian embryos, free from other components contained in poultry eggs or non-human mammalian embryos, and uncontaminated by exogenous substances. Pure amniotic fluid can be stored in a refrigerator at -60°C or below and used after thawing.

[0024] The amniotic fluid described herein can be used as an active ingredient in a pharmaceutical agent for administration to a subject in need thereof to promote cell proliferation and tissue repair in the body. For example, an effective amount of the amniotic fluid or a pharmaceutical composition containing amniotic fluid described herein can be administered to a subject in need thereof.

[0025] Combining CN 201810911038.2, CN 201810909485.4, CN 201810909193.0 and CN 201910887556.X, it should be understood that amniotic fluid from various sources described in the present invention exhibits the same or similar biological activities, and when used in the present invention, all can modulate the TLR4 / NF-κB signaling pathway, inhibit M1 macrophages, and increase M2 macrophages, thereby being usable for the treatment or prevention of diseases that benefit from modulation of the TLR4 / NF-κB signaling pathway and macrophage-mediated diseases.

[0026] As used herein, animals are mammals, particularly humans.

[0027] disease Toll-like receptors (TLRs) are a family of receptors that mediate innate immunity. TLR4, in particular, can recognize various pathogen-associated molecular patterns (PAMPs) and activate inflammatory cells. TLR4 recognizes not only the exogenous ligand lipopolysaccharide (LPS) but also endogenous ligands expressed during arterial injury, and regulates pathological processes such as cholesterol metabolism, plate stability, cell apoptosis, inflammation, vascular remodeling, and immune responses. TLR binding to its ligand ultimately leads to the activation of NF-κB, which regulates the expression of inflammation- and immune-related genes.

[0028] NF-κB can play a role at multiple stages in disease development, including inflammatory responses, foam cell formation, vascular smooth muscle proliferation, and cell apoptosis. It regulates enzymes (e.g., cyclooxygenase 2) involved in early low-density lipoprotein (LDL) modification and inflammatory lipid formation. Activated NF-κB can also promote the transcription of factors such as adhesion molecules, inflammatory cytokines, chemokines, and growth factors, which play important roles in the development and progression of inflammatory diseases.

[0029] In this specification, TLR4 / NF-κB signaling pathway-mediated diseases include systemic lupus erythematosus (Ji Juan. The role of bone marrow mesenchymal stem cell senescence caused by abnormal activation of TLR4 signaling in the development of systemic lupus erythematosus [D]. Nantong University, 2017.), atherosclerosis (Li Hongmei, Wang Xian. Research progress on the relationship between TLR4 / MyD88 / NF-κB signaling pathway and atherosclerotic cardiovascular disease [J]. Chinese Journal of Cardiovascular Medicine, 2017, 9(09):1132-1134.), and coronary artery disease (Lin Yongjun et al. The effect of Ginkgo Biloba Drop Pill on TLR4 in patients with coronary artery disease). Effects on the TLR4 / NF-κB signaling pathway and immune indicators [J]. Straits Pharmacology, 2017, 29(03):89-92.), vascular inflammation, myocardial ischemic tissue inflammation (Zhou Xueling. Investigation of the mechanism of action of Huoxin Wan on acute myocardial ischemic inflammatory responses via the TLR4 / NF-κB signaling pathway [D]. Fujian University of Traditional Chinese Medicine, 2020. DOI:10.27021 / d.cnki.gfjzc.202020.000334.), myocardial injury (Wei Hao et al. Analysis of the protective mechanism of HIF-1α against myocardial injury in rats after myocardial ischemia / reperfusion via the TLR4 / NF-κB signaling pathway [J]. .Journal of Clinical and Experimental Medicine, 2019,18(10):1017-1020.), myocarditis, liver failure (Liu Qiaohong, Liu Jiangkai, Li Suling. Therapeutic effect of detoxifying yutongfu granules on patients with subacute, chronic and acute liver failure and its effect on the TLR4 / NF-κB inflammatory pathway [J]. Journal of Integrated Chinese and Western Medicine for Liver Diseases, 2018,28(05):264-267.), alcoholic liver damage (Zhu Xiaoning, Wang Jing, Zhang Yurong, Yin Gezu, Peng Mengyun, Zeng Yong. Expectorant and blood-activating prescription is SOC Inhibiting the TLR4 / NF-κB signaling pathway through upregulation of S1 ameliorates liver injury in mice with non-alcoholic steatohepatitis [J]. World Science and Technology of Traditional Chinese Medicine Modernization, 2020, 22(12):4293-4299.), inflammatory immune responses during alcohol metabolism (Yin Xiaolei, Lu Weina, Feng Liying. The role of the LPS / TLR4 signaling pathway in non-alcoholic fatty liver disease. World Chinese Journal of Digestion, 2013, 21(28):2957-2962. DOI:10.11569 / wcjd.v21.i28.2957).World Chinese Journal of Digestive Diseases 2013,21(28): 2957-2962. DOI:10.11569 / wcjd.v21.i28.2957), acute lung injury (Niu Zequn, Wang Liming, Feng Hui, Sun Jiangli, Pei Honghong, Pan Longfei. Study on the role of TLR4 / NF-κB signaling pathway in acute severe pancreatitis-associated lung injury in rats [J]. Modern Digestive and Interventional Medicine, 2019,24(12):1404-1407.), chronic obstructive pulmonary disease (Chen Xunchun, Li Minglan, Pan Biyun, Wang Yanying, Ding Yipeng. LnCRNA via TLR4 / NF-κB signaling pathway Control of airway inflammation and remodeling in chronic obstructive pulmonary disease through activation of RP11-20G6 [J]. Bulletin of Anhui Medical University, 2022, 57(4):586-593.), pneumonia such as silicosis (Zhu Lili. Study on the role of inflammatory factors mediated by TLR4 and RAGE in the pathogenesis of silicosis [D]. Shanxi Medical University, 2014.), acute kidney injury (Zhan Yun, Zhang Yingjie, Leng Bin. Astragaloside IV activates TLR Improves lipopolysaccharide-induced acute vascular endothelial injury via the HMGB1 / TLR / NF-κB pathway [J]. Pharmacology and Clinical Use of Traditional Chinese Medicine, 2018, 34(03):77-80. DOI:10.13412 / j.cnki.zyyl.2018.03.019.), lupus nephritis (Chen Ning. Expression and significance of the HMGB1 / TLR / NF-κB signaling pathway in the kidney tissue of lupus nephritis mice [D]. Hebei Medical University, 2010.). Nephritis, acute enteritis (Li Xuehui et al., Study on the effect and mechanism of CD11b agonist leukadherin-1 on the development of experimental colitis in mice [J]. Chinese Journal of Microbiology and Immunology, 2019(12):904-905-906-907-908-909-910.), ulcerative colitis (Lin Xiaoyuan, Liu Jiemin, TLR4 / MyD88 / NF-κB signaling pathway and ulcerative colitis [J]. Gastrointestinal Pathology, 2013,18(04):244-246.), inflammatory bowel diseases such as radiation proctitis (Zhu Chaofu, An Baiping, Huang Hongjie, Du Chi, Wu Yongjun, Lan Lan, Li Dan, Lei Dongmei, Li Shijie, Ao Rui. Investigation of the mechanism of action of Toli Disinfecting Powder in the treatment of radiation proctitis based on the TLR4 / NF-κB signaling pathway [J]. Bulletin of Anhui Medical University, 2020,55(09):1367-1373. DOI:10.19405 / j.cnki.issn 1000-1492.2020.09.010.), and chronic atrophic gastritis (Zhou Wei, Yuan Xing.Effect of American cockroach extract on TLR4 / NF-κB signaling pathway in rats with chronic atrophic gastritis [J]. World Chinese Journal of Digestion, 2017, 25(21):1945-1951.), gastritis such as pneumonia (Guo Shasha. Role of TLR-MYD88-NF-κBBP65-dependent signaling pathway in lung tissue of mice with Mycoplasma pneumonia [D]. Qingdao University, 2017.), bronchitis (Wu Ning, Huang Yuxiao, Shi Xue, Chen Jinlun, Peng Lingfeng, Yang Lulu, Xu Hong, Sun Jianfei, Liu Hua. Effect of the Miao medicinal herb "Guan Nu Zin Yan" on TLR4-MyD88-NF-κB signaling pathway in rats with chronic bronchitis [J]. Guizhou Medical College Bulletin of Nanjing University of Chinese Medicine, 2020, 45(11): 1283-1288. DOI: 10.9367 / j.cnki.2096-8388.2011.009.), pharyngitis (Xu Jinhong. Study on the mechanism of Zizhengdihuangtang on acute pharyngitis based on the TLR4 / NF-κB signaling pathway [D]. Anhui University of Traditional Chinese Medicine, 2020. DOI: 10.26922 / d.cnki.ganzc.202020.000216.), sinusitis (Xiao Jianying, Xue Shanshan, Ni Pingmin, Wang Zhuo, Wu Youjun. Study on the mechanism of action of Naiyuan combination on acute sinusitis based on the NF-κB signaling pathway [J]. Nanjing University of Traditional Chinese Medicine Bulletin, 2022, 38(3): 247-253. doi: 10.14148 / j.issn.1672-0482.2022.0247), acute respiratory infections such as otitis media (CN201180025442.6, Luo Luncai, Tong Yan, Zhang Xingguo et al. Study on the anti-otitis media effect of Heiyu capsule based on the TLR4 / MyD88 / NF-κB pathway [J]. Northwest Pharmaceutical Journal, 2021, 36(01):66-71.), periodontitis, hyperuricemia (Guo Zhuling, Tang Han, Huang Miao et al. TLR4 / NF-κB signaling in the regulation of periodontitis and hyperuricemia and their interactions) Advances in the study of the pathway of quercetin delivery [J]. Chinese Journal of Stomatology Research: Electronic Edition, 2021, 15(1):5.), asthma (Li Hongjia. Study on the mechanism by which quercetin regulates airway inflammation in asthmatic patients via TLR4 / NF-κB signaling [D]. Shandong University, 2015.), allergic rhinitis (Yin Lili. Expression and mechanism of action of TREM-1, TLR4, TNF-α and NF-κB in the nasal mucosa of mice with allergic rhinitis [D]. Huazhong University of Science and Technology, 2013.), hypersensitive rhinitis (Hu Chen, Liang Chenyang, Zhou Weiguo.Expression and role of TLR4 / NF-κB pathway mediated by inflammation in patients with hypersensitive rhinitis [J]. Labeling Immunoanalysis and Clinical, 2018, 25(06):788-790+838.), mastitis (LU Jinye, GU Baibai. Research advances in TLR4 / My D88 / NF-κB signaling pathway and mastitis [J]. Animal Husbandry and Veterinary Medicine, 2018, 50(11):127-129.), acute gouty arthritis (LUO Fei, MEI Yan. Effects of procyanidins on TLR4 / NF-κB signaling pathway in rats with acute gouty arthritis. Chinese Journal of Clinical Pharmacology and Therapeutics. 2018, (1): 41-46. DOI: 10.12092 / j.issn.1009-2501.2018.01.008), chronic arthritis (Yu Bijun. Role of TLR signaling pathway in chronic inflammatory arthritis [J]. International Journal of Immunology, 2007, 30(06):443-447.), rheumatoid arthritis (Bai Lin, Yang Yuxin, Wan Qiaofeng, et al. Baicalin alleviates synovitis in rats with rheumatoid arthritis via the TLR2 / NF-κB pathway [J]. Chinese Pharmacology Bulletin, 2017, 33(11):1569-73.), inflammation of wound tissue, hypertrophic scars (Li Qiang. TRAIL, DR5, and NF-κB in refractory wound granulation tissue and hypertrophic scars in humans. Study on the expression and significance of p65 [D]. Shandong University, 2008.), polycystic ovary syndrome (Yao Zhilin, Huang Yinghong, Xu Xiaojuan. Effect of Kidney-Treating Phlegm Formula on TLR4 / NF-κB inflammatory signaling pathway, oxidative stress, and insulin resistance in female mice with polycystic ovary syndrome [J]. Sichuan Journal of Traditional Chinese Medicine, 2019, 37(11):6.), pituitary prolactin adenoma (Guo Runzhu. Study on the pharmacodynamic effects and mechanisms of hordenine on hyperprolactinemia and prolactinoma [D]. Hubei University of Traditional Chinese Medicine, 2019. DOI:10.27134 / d.cnki.ghbzc.2019.00015.), adrenocorticotropic hormone adenoma (Wu Q, Feng Y, Liu L, Liu Y, Liu X, Zhang L, Li Y, Wang L.Corticotropin-Releasing Factor Aggravates Ischemic Stroke Injury by the Inflammatory Activation of Microglia. Endocrinology. 2022 Mar 1;163(3):bqac013. doi: 10.1210 / endocr / bqac013. PMID: 35137012.), intracranial aneurysm (Wang Y, Jin J. Roles of macrophages in formation and progression of intracranial aneurysms. Zhejiang Da Xue Xue Bao Yi Xue Ban. 2019 Apr 25;48(2):204-213. Chinese. doi: 10.3785 / j.issn.1008-9292.2019.04.13. PMID: 31309760; PMCID: PMC8800668.), infectious diseases such as bacterial infection, fungal infection, and viral infection (Jianli Yan, Hongyu Cheng, Wenluo, Guoyue Zhong, and Jixiao Zhu. Research progress on the mechanism of TLR / MyD 88 / NF-κB signaling pathway in various diseases [J]. Bulletin of Chinese Pharmacology, 2019, 35(4):451-455), allergic skin diseases, bronchial asthma, and hypersensitivity rhinitis (Jianli Yan, Hongyu Cheng, Wenluo, Guoyue Zhong, and Jixiao Zhu. Research progress on the mechanism of TLR / MyD 88 / NF-κB signaling pathway in various diseases [J]. Bulletin of Chinese Pharmacology, 2019, 35(4):451-455). These include, but are not limited to, inflammatory responses caused by neurological and / or vascular disorders, selected from allergic diseases such as TLR9, MyD88, and NF-κB signaling pathway (Progress in research on the mechanism of TLR9 / NF-κB signaling pathway [J]. Chinese Pharmacology Bulletin, 2019, 35(4):451-455), and allergic purpura (Wang Wang, Yang Lijun. Expression and significance of TLR9, MyD88, and NF-κB in allergic purpura [J]. Chinese Modern Physician, 2016, 54(29):9-12).

[0030] The amniotic fluid described herein can treat or prevent various TLR4 / NF-κB signaling pathway-mediated diseases by suppressing activation of the TLR4 / NF-κB signaling pathway.

[0031] In this specification, macrophage-mediated refers to the dynamic balance and imbalance of cell subgroups with different functional characteristics within macrophages and their role in the pathological process of disease. Macrophage-mediated diseases include hypertrophic scars (Li Zhenjiang, Li Shujun, Zhou Jian et al. Study on macrophage activation-related factors in hypertrophic scar tissue at different times [J]. Journal of Zunyi Medical University, 2022, 45(1):87-91), chronic obstructive pulmonary disease (Kei Shigang. Study on the role of macrophages in the pathogenesis of chronic obstructive pulmonary disease [J]. Chinese Practical Medicine, 2019, 14(12):196-197), tumors such as breast cancer and liver cancer, metabolic diseases such as severe obesity, insulin resistance, and type 2 diabetes, acute pancreatitis, and vascular diseases. Inflammatory diseases such as atherosclerosis (Wang Yongkang, Li Jiayi, Guan Fei, Lei Jiahui. Macrophage polarization mechanism and its role in common diseases [J]. Tropical Diseases and Parasitology, 2022, 20(2): 103-108, 112), cardiovascular diseases such as myocarditis, myocardial infarction, and arrhythmia (Dong Wei and Miao Liu. Research advances in the function of macrophages and their role in heart diseases [J]. Modern Chinese Medicine, 2022, 29(16): 49-52), neurological disorders such as Alzheimer's disease, and cerebral infarction (Wang Jiahui. Introduction: Macrophages and related diseases Research progress on [J]. Practical Geriatrics, 2021, 35(12):1217-1218), brain injury (Zhou Zhongji, Lei Rui, Yin Shi, Liu Hongling, Liu Kei. Correlation analysis between M1 macrophage levels and the severity and prognosis of coronary artery disease [J]. Chinese Medicine Innovation, 2022, 19(4):161-165), autoimmune uveitis, retinopathy, keratitis, corneal transplantation, Sjögren's syndrome, uveal melanoma, myopia (Qu Ruyi, Zhou Mengxian, Bi Hongsheng, Guo Dadong. Mechanism of macrophage polarization in the development and progression of ocular diseases. Research progress on the relationship between macrophage M1 / M2 polarization and immune inflammatory diseases [J]. Chinese Journal of Immunology, 2021, 37(22):2791-2797), alcoholic liver disease, colitis (Wu Yan, Zhang Dingran, Wang Xinhui, Xu Hongyang, Liu Peiyao, Qi Zhili. Research progress on macrophage polarization and its role in inflammatory diseases [J]. New Progress in Ophthalmology, 2022, 42(3):239-243), eye diseases such as inflammatory bowel disease, autoimmune hepatitis, asthma (Jia Rui, Hui Yi, Yan Shuguang (director), Li Jingtao. Research progress on the relationship between macrophage M1 / M2 polarization and immune inflammatory diseases [J]. Chinese Journal of Immunology, 2021, 37(22):2791-2797), alcoholic liver disease, colitis (Wu Yan, Zhang Dingran, Wang Xinhui, Xu Hongyang, Liu Peiyao, Qi Zhili. Research progress on macrophage polarization and its role in inflammatory diseases [J].Chinese Journal of Animal Husbandry, 2021, 57(7):22-26), multiple sclerosis (Li Xing, Wang Dandan, Tang Qi, Liu Jie, Gu Zhongyi, Zhao Huan, Sun Hongchen. Research advances in transcriptional regulation of macrophage polarization and its impact on related diseases [J]. Journal of Jilin University: Medical Edition, 2016, 0(3):622-625), periodontitis (Zhou Qi, Sun Huijuan, Yu Donghua, Liu Shumin. Mechanism of action of macrophage M1 / M2 polarization in various diseases [J]. Chinese Pharmacological Bulletin, 2020, 36(11):1502-1506), etc. Immunoinflammatory diseases, such as rheumatoid arthritis, bone erosion, synovitis (Wang Dongyi, Shen Junyi, Lu Le, Cai Hui. Correlation between macrophage polarization imbalance and disease activity and bone erosion in rheumatoid arthritis [J]. Journal of Graduate Medical Sciences, 2021, 34(8):823-828), osteoarthritis (Zhou Qi, Sun Huijuan, Yu Donghua, Liu Shumin. Mechanism of macrophage M1 / M2 polarization in various diseases [J]. Chinese Pharmacology Bulletin, 2020, 36(11):1502-1506), arthritis, acute kidney injury, chronic Kidney disease, end-stage renal failure, proliferative glomerulonephritis, membranous nephropathy, diabetic nephropathy, purpura nephritis, ANCA-associated small-vessel vasculitis, urinary tract infection, autosomal dominant polycystic kidney disease (Yu Na, Wang Bo, Huang Pintao, Hak Jian. Research progress on macrophage migration inhibitory factor in the pathogenesis of nephritic diseases [J]. Hebei Medicine, 2021, 27(1):170-174), and other nephritis, sepsis (Huang Xuechao, Shen Shiyang, Mo Ran. Research progress on macrophage membrane biomimetic nanodrug delivery systems for the treatment of inflammatory diseases [J]). These include, but are not limited to, bacterial infections such as gestational hypertension (Zhao Cai-zhen, Qiao Fu-yuan. Macrophages and Pregnancy-Related Hypertensive Disease [J]. Chinese Journal of Eugenics and Genetics, 2006, 14(3):126-128), diabetes, gestational diabetes, and diabetic nephropathy (Zhou Qi, Sun Hui-juan, Yu Dong-hua, Liu Shu-min. Mechanism of Macrophage M1 / M2 Polarization in Various Diseases [J]. Chinese Pharmacological Bulletin, 2020, 36(11):1502-1506).

[0032] There are two subtypes of macrophages: M1 and M2. M1 macrophages, also known as pro-inflammatory macrophages, have the primary function in vivo of phagocytosing endogenous substances, such as bacteria and apoptotic cell debris, thereby protecting tissues and organs from foreign invasion. In inflammatory diseases, M1 macrophages accumulate at early inflammatory sites, are activated by inflammatory cytokines, and secrete inflammatory factors to promote the onset and progression of inflammation and protect the body from foreign invasion. In contrast, in the later stages of inflammation, M2 macrophages play roles in inflammation suppression, tissue repair, and tissue structural reconstruction, and are therefore also known as anti-inflammatory or immunoregulatory macrophages. The ratio of M1 / M2 macrophage populations during the onset and progression of inflammation continuously changes over time, eventually resulting in complete resolution of the effects of inflammation.

[0033] In this specification, M1 macrophage-mediated refers to the effects of M1 macrophages in diseases, such as being activated by inflammatory cytokines, secreting inflammatory factors, and protecting the body from foreign invasion. M1 macrophage-mediated diseases include involutional hypertrophic scars (Li Zhenjiang, Li Shujun, Zhou Jian et al., Study on macrophage activation-related factors in hypertrophic scar tissue at different stages [J], Journal of Zunyi Medical University, 2022, 45(1):87-91), chronic obstructive pulmonary disease (Kei Shigang, Study on the role of macrophages in the pathogenesis of chronic obstructive pulmonary disease [J], Chinese Practical Medicine, 2019, 14(12):196-197), breast cancer, liver cancer, etc. Metabolic diseases such as tumors, severe obesity, insulin resistance, and type 2 diabetes, inflammatory diseases such as acute pancreatitis (Wang Yongkang, Li Jiayi, Guan Fei, Lei Jiahui. Macrophage polarization mechanism and its role in common diseases [J]. Tropical Diseases and Parasitology, 2022, 20(2):103-108, 112), coronary artery disease such as atherosclerosis, kidney disease, and obesity (Zhou Zhongji, Lei Rui, Yin Shi, Liu Hongling, Liu Kei. M1 macrophage levels and the severity of coronary artery disease and Correlation analysis with prognosis [J]. Chinese Medicine Innovation, 2022, 19(4): 161-165), cardiovascular diseases such as myocarditis and myocardial infarction (Dong Wei, Miao Liu. Research progress on the function of macrophages and their role in heart disease [J]. Modern Chinese Medicine, 2022, 29(16): 49-52), and cerebral infarction (Wang Jiahe. Introduction: Research progress on macrophages and related diseases [J]. Practical Geriatrics, 2021, 35(12): 1217-1218). Brain diseases such as brain injury (Zhou Zhongji, Lei Rui, Yin Shi, Liu Hongling, Liu Kei. Correlation analysis between M1 macrophage levels and the severity and prognosis of coronary artery disease [J]. Chinese Medical Innovation, 2022, 19(4):161-165), autoimmune uveitis, retinopathy, keratitis, corneal transplantation, Sjögren's syndrome, and uveal melanoma (Qu Ruyi, Zhou Mengxian, Bi Hongsheng, Guo Dadong. Research progress on the mechanism of macrophage polarization in the development and progression of ocular diseases [J]).New Progress in Ophthalmology, 2022,42(3):239-243), eye diseases such as inflammatory bowel disease, autoimmune hepatitis, asthma (Jia Rui, Hui Yi, Yan Shuguang (director), Li Jingtao. Research progress on the relationship between macrophage M1 / M2 polarization and immune inflammatory diseases [J]. Chinese Journal of Immunology, 2021,37(22):2791-2797), alcoholic liver disease, colitis (Wu Yan, Zhang Dingran, Wang Xinhui, Xu Hongyang, Liu Peiyao, Qi Zhili. Research progress on macrophage polarization and its effect on inflammatory diseases [J]. Chinese Journal of Animal Husbandry, 2021,57(7):22- 26), multiple sclerosis (Li Xing, Wang Dandan, Tang Qi, Liu Jie, Gu Zhongyi, Zhao Huan, Sun Hongchen. Research advances in the transcriptional regulation of macrophage polarization and its impact on related diseases [J]. Journal of Jilin University: Medical Edition, 2016,0(3):622-625), immune-inflammatory diseases such as periodontitis and osteoarthritis, diabetes, gestational diabetes, and diabetic nephropathy (Zhou Qi, Sun Huijuan, Yu Donghua, Liu Shumin. Mechanism of action of macrophage M1 / M2 polarization in various diseases [J]. Bulletin of Chinese Pharmacology, 2020,36(11):1502-1506).

[0034] In some embodiments, M1 macrophage-mediated diseases include, but are not limited to, immune inflammatory diseases, metabolic diseases, diabetes, and tumors. In an exemplary embodiment, the M1 macrophage-mediated disease described herein is persistent chronic inflammation in diabetic wounds.

[0035] In this specification, M2 macrophage-mediated disease refers to the effects of M2 macrophages in disease, such as suppressing inflammation and inflammatory factors and promoting wound healing and repair. M2 macrophage-mediated diseases include inflammatory diseases such as proliferative hypertrophic scars (Li Zhenjiang, Li Shujun, Zhou Jian, et al., Study on macrophage activation-related factors in hypertrophic scar tissue at different stages [J]. Journal of Zunyi Medical University, 2022, 45(1):87-91), tumors such as breast cancer and liver cancer, metabolic diseases such as insulin resistance and type 2 diabetes, acute pancreatitis (Wang Yongkang, Li Jiayi, Guan Fei, Lei Jiahui. Macrophage polarization mechanisms and their role in common diseases [J]. Tropical Diseases and Parasitology, 2022, 20(2):103-108, 112), myocardial infarction, myocardial failure, atherosclerosis, and coronary artery disease (Zhang Xiangning, Dang Guohui, Feng Juan (guidance)). Regulation of exosomes on M2 macrophages and its role in cardiovascular diseases [J]. Chinese Journal of Immunology, 2022,38(10):1257-1262), myocarditis (Dong Wei, Miao Liu. Research progress on the function of macrophages and its role in heart disease [J]. Modern Chinese Medicine, 2022,29(16):49-52), cerebral infarction (Wang Jiahe. Introduction: Research progress on macrophages and related diseases [J]. Practical Geriatrics, 2021,35(12):1217-1218), autoimmune uveitis, retinopathy, keratitis, corneal transplantation, Sjögren's syndrome, and uveal melanoma (Qu Ruyi, Zhou Mengxian, Bi Hongsheng, Guo Dadong. Research progress on the mechanism of macrophage polarization in the development and progression of ocular diseases [J].New Progress in Ophthalmology, 2022,42(3):239-243), eye diseases such as inflammatory bowel disease, autoimmune hepatitis, asthma (Jia Rui, Hui Yi, Yan Shuguang (director), Li Jingtao. Research progress on the relationship between macrophage M1 / M2 polarization and immune inflammatory diseases [J]. Chinese Journal of Immunology, 2021,37(22):2791-2797), alcoholic liver disease, colitis (Wu Yan, Zhang Dingran, Wang Xinhui, Xu Hongyang, Liu Peiyao, Qi Zhili. Research progress on macrophage polarization and its effect on inflammatory diseases [J]. Chinese Journal of Animal Husbandry, 2021,57(7):22- 26), multiple sclerosis (Li Xing, Wang Dandan, Tang Qi, Liu Jie, Gu Zhongyi, Zhao Huan, Sun Hongchen. Research advances in the transcriptional regulation of macrophage polarization and its impact on related diseases [J]. Journal of Jilin University: Medical Edition, 2016,0(3):622-625), immune-inflammatory diseases such as periodontitis and osteoarthritis, diabetes, gestational diabetes, and diabetic nephropathy (Zhou Qi, Sun Huijuan, Yu Donghua, Liu Shumin. Mechanism of action of macrophage M1 / M2 polarization in various diseases [J]. Bulletin of Chinese Pharmacology, 2020,36(11):1502-1506).

[0036] In some embodiments, M2 macrophage-mediated diseases include, but are not limited to, immune inflammatory diseases, metabolic diseases, diabetes, and tumors. In an exemplary embodiment, the M2 macrophage-mediated disease described herein is persistent chronic inflammation in diabetic wounds.

[0037] In some embodiments, the amniotic fluid described herein treats and prevents macrophage-mediated diseases by decreasing the number of M1 macrophages and increasing the proportion of M2 macrophages.

[0038] In a particularly preferred embodiment of the present invention, amniotic fluid, particularly amniotic fluid from poultry eggs as described herein, more preferably from chicken eggs, is used to prevent or treat macrophage-mediated diseases.

[0039] Pharmaceutical Composition The present specification also provides a pharmaceutical composition comprising the amniotic fluid described herein, particularly amniotic fluid from poultry eggs, more preferably amniotic fluid from chicken eggs at embryonic days 5 to 12, more preferably 6 to 11, more preferably 6 to 9, and even more preferably 7 to 8. The pharmaceutical composition may be amniotic fluid stored frozen at -60°C or below, or a freeze-dried reagent thereof, such as freeze-dried amniotic fluid. The pharmaceutical composition may also contain other pharmaceutically acceptable carriers or excipients, such as injectable saline, injectable water, or glucose injection solution. Preferably, the pharmaceutical composition contains 5 to 40% (v / v) or 10 to 35%, preferably 15 to 30%, of amniotic fluid.

[0040] Pharmaceutical compositions containing amniotic fluid described herein generally also contain pharmaceutically acceptable additives. As used herein, the term "pharmaceutically acceptable additive" refers to a carrier, diluent, and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and includes, but is not limited to, antibiotics, humectants, pH adjusters, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, preservatives, carriers, flow aids, sweeteners, dyes / colorants, flavor enhancers, wetting agents, dispersants, suspending agents, stabilizers, isotonicity agents, solvents, or emulsifiers. In some embodiments, pharmaceutically acceptable additives can include one or more inactive ingredients, including, but not limited to, stabilizers, preservatives, additives, adjuvants, propellants, compressed air or other suitable gases, or other inactive ingredients suitable for use in combination with the therapeutic compound. More specifically, suitable pharmaceutically acceptable additives may be additives commonly used in the art for diabetic foot disease. In one or more embodiments, suitable pharmaceutically acceptable additives for sprays are selected from one or more of water, gluconolactone, sodium benzoate, arbutin, sodium hyaluronate, nicotinamide, and glycerin.In one or more embodiments, suitable pharmaceutically acceptable additives for coatings or dressings are selected from one or more of water, glycerin, panthenol, magnesium ascorbyl phosphate, nicotinamide, sodium hyaluronate, phenoxyethanol, caprylyl glycol, and sorbic acid.Those skilled in the art can determine the content of pharmaceutically acceptable additives according to actual circumstances.

[0041] When a medication is sprayed, painted, or applied to a wound, a dressing is usually required. A "dressing" is a wound wrap or material used to cover a sore, cut, or other lesion. Types of wound dressings include passive dressings, interactive dressings, and bioactive dressings. Wound dressings suitable for use in the present invention are known in the art.

[0042] In addition to amniotic fluid, the pharmaceutical compositions described herein may contain other active ingredients that contribute to wound healing in a patient, including, but not limited to, Centella asiatica extract, rose dew, licorice root extract, olive leaf extract, marigold flower extract, white willow bark extract, lavender extract, lemon fruit extract, hydrolyzed soy protein, artemisia princeps extract, tea leaf artemisia extract, thyme extract, Echinacea purpurea extract, St. John's wort flower / leaf extract, aloe vera leaf juice powder, and yeast extract.

[0043] Purpose of use and treatment method Also provided herein is the use of amniotic fluid described in any of the embodiments herein in the manufacture of a formulation for treating and / or preventing a TLR4 / NF-κB signaling pathway-mediated disease. Another aspect of the present specification also provides the use of amniotic fluid described in any of the embodiments herein in the manufacture of a formulation for treating and / or preventing a macrophage-mediated disease. Another aspect of the present specification also provides the use of amniotic fluid described in any of the embodiments herein in the manufacture of a formulation for treating and / or preventing an M1 macrophage-mediated disease. Another aspect of the present specification also provides the use of amniotic fluid described in any of the embodiments herein in the manufacture of a formulation for treating and / or preventing an M2 macrophage-mediated disease. Another aspect of the present specification also provides the use of amniotic fluid described in any of the embodiments herein in the manufacture of a formulation for suppressing the number of M1 macrophage populations and increasing the proportion of M2 macrophage species. Another aspect of the present specification also provides the use of amniotic fluid described in any of the embodiments herein in the manufacture of a formulation for promoting the transformation of M1 macrophages to M2 macrophages.

[0044] In some embodiments, the present invention provides use of the amniotic fluid described in any of the embodiments herein in the preparation of a reagent for one or more of the following purposes: (1) improving the rate of wound healing in a patient; (2) promoting the growth of new blood vessels in a patient's wound; (3) reducing the inflammatory cytokines IL-6 and TNF-α and increasing TGF-β1 and IL-10 in a patient with inflammation; (4) promoting the formation of type III collagen in a patient's wound or increasing the ratio of type III collagen to type I collagen in a patient's wound; and (5) reducing the transcription and translation levels of iNOS, TNF-α, IL-6, and IL-1β and increasing the transcription and translation levels of CD206 and Arg-1 in a patient with inflammation.

[0045] In some embodiments, the present specification also provides amniotic fluid or a composition comprising amniotic fluid as described in any of the embodiments herein for treating and / or preventing a TLR4 / NF-κB signaling pathway-mediated disease, treating and / or preventing a macrophage-mediated disease, treating and / or preventing an M1 macrophage-mediated disease, treating and / or preventing an M2 macrophage-mediated disease, suppressing the number of M1 macrophage populations, increasing the proportion of M2 macrophage types, and / or promoting the transformation of M1 macrophages to M2 macrophages.

[0046] Also provided herein are methods for treating and / or preventing a TLR4 / NF-κB signaling pathway-mediated disease, comprising administering to a subject in need thereof a therapeutically effective amount of amniotic fluid or a composition comprising amniotic fluid as described herein.

[0047] The present invention also provides methods for treating and / or preventing macrophage-mediated diseases. The present invention also provides methods for treating and / or preventing M1 macrophage-mediated diseases. The present invention also provides methods for treating and / or preventing M2 macrophage-mediated diseases. These methods include administering to a subject in need thereof a therapeutically effective amount of amniotic fluid or a composition comprising amniotic fluid as described herein.

[0048] The present invention also provides a method for promoting the transformation of M1 macrophages to M2 macrophages, comprising administering to a subject in need thereof an effective amount of amniotic fluid or a composition comprising amniotic fluid as described herein. In some embodiments, the method is used to treat or prevent a disease that would benefit from an increase in M2 macrophages, including, but not limited to, the various diseases described above. The method may be an in vivo method or an in vitro method.

[0049] The present invention also provides a method for tissue repair in an inflammatory subject, the method comprising culturing target tissue cells in vitro using the amniotic fluid described herein, or a composite dressing prepared using amniotic fluid as a main ingredient, or a cell culture medium containing the amniotic fluid described herein to form a tissue matrix, and then implanting the tissue matrix into the site of tissue damage or defect.

[0050] The present specification also provides a method for repairing inflamed tissue, which includes culturing tissue cells of interest in vitro using the amniotic fluid described herein or a composite dressing prepared using the amniotic fluid as a main ingredient, or a cell culture medium containing the amniotic fluid described herein to form a tissue matrix, and then transplanting the tissue matrix into a site of tissue damage or defect.

[0051] In some embodiments, the present disclosure also provides a method for improving the rate of wound healing in a patient, comprising applying amniotic fluid or a pharmaceutical composition containing the amniotic fluid described herein to the patient's wound or applying a composite dressing prepared primarily using amniotic fluid of the present invention. In some embodiments, the present disclosure also provides a method for promoting the growth of new blood vessels in a patient's wound, comprising applying amniotic fluid or a pharmaceutical composition containing the amniotic fluid described herein to the patient's wound or applying a composite dressing prepared primarily using amniotic fluid of the present invention. In some embodiments, the present disclosure also provides a method for reducing the pro-inflammatory cytokines IL-6 and TNF-α and increasing TGF-β1 and IL-10 in a patient with inflammation, comprising administering to the patient an effective amount of amniotic fluid or a pharmaceutical composition containing the amniotic fluid described herein. In some embodiments, the present disclosure also provides a method for promoting the formation of type III collagen in a patient's wound or increasing the ratio of type III collagen to type I collagen in a patient's wound, comprising applying amniotic fluid or a pharmaceutical composition containing the amniotic fluid described herein to the patient's wound or applying a composite dressing prepared primarily using amniotic fluid of the present invention. In some embodiments, the present invention also provides methods for reducing the transcription and translation levels of iNOS, TNF-α, IL-6, and IL-1β, and increasing the transcription and translation levels of CD206 and Arg-1 in a patient with inflammation, comprising administering to the patient an effective amount of amniotic fluid or a pharmaceutical composition comprising amniotic fluid as described herein.

[0052] As used herein, a therapeutically effective amount refers to an amount that is capable of achieving treatment, prevention, relief and / or alleviation of a disease or symptom in a subject.

[0053] The therapeutically effective amount can be determined depending on factors such as the patient's age, sex, existing disease and its severity, other physical conditions of the patient, etc. As used herein, a subject or patient generally refers to a mammal, particularly a human.

[0054] As used herein, the dosage and frequency of administration can be determined by a physician depending on the specific condition and the patient's age and gender. Generally, in the treatment of a specific disease, a therapeutically effective amount refers to an amount sufficient to improve or in some way alleviate symptoms associated with the disease. Such a dosage can be administered as a single dose or according to an effective treatment plan. Drug dosages are generally administered to improve symptoms, although they may also cure the disease. Repeated administration is generally required to achieve the desired improvement in symptoms. For example, a human dosage can typically be 1 to 200 mL per injection, administered daily or weekly. In certain embodiments, the administration frequency is several times a day, twice a day, every 2 days, 3 days, 4 days, 5 days, or 6 days, or once every two or three days or once a month.

[0055] Cell culture medium In certain embodiments, the present specification also provides a cell culture medium containing an appropriate amount of amniotic fluid described herein. The content of amniotic fluid in the cell culture medium can be determined depending on the type of cell being cultured. For example, the amount of amniotic fluid added may be 0.1 to 30% by weight of the cell culture medium, such as 1 to 25% or 3 to 20%. An appropriate cell culture medium can be selected depending on the cells being cultured. Exemplary cell culture media include, but are not limited to, various commercially available media such as DMEM, RPMI 1640, MEM, and DMEM / F12.

[0056] The present invention will be further described in the following specific examples. It should be understood that these examples are merely illustrative and do not limit the scope of the present invention. Unless otherwise specified, the methods and reagents used in the examples are conventional methods and reagents in the art.

[0057] Materials and Methods Antibodies and reagents PE CD206 antibody (12-2069-42) and FITC F4 / 80 antibody (11-4801-82) were purchased from eBioscience. Arginase 1 (Arg-1; 93668), α-SMA (19245), and CD206 (24595) were purchased from CST. GAPDH (ab181602), iNOS (ab178945), TNF-α (ab183218), IL-6 (ab290735), IL-1β (ab254360), and CD31 (ab281583) were purchased from Abcam. TLR4 (A5258), NF-κB-p65 (A19653), and p-IKB (AP0707) were purchased from ABclonal. Trizol reagent and SYBR green were purchased from Vazyme Biotech. STZ (S0130) and glucose (D9434) were purchased from Sigma-Aldrich.

[0058] ceAF production Fertilized eggs were incubated at 38°C and 50% humidity. ceAF was isolated between days 6 and 8 of incubation. After centrifugation (2500 × g, 20 min), the supernatant was filtered through a 0.22 μm sterile filter (Millipore, USA), and the filtered sample was aliquoted and stored at −80°C.

[0059] Cell culture RAW264.7 cells were provided by the Cell Bank of the Chinese Academy of Sciences and cultured in DMEM high-glucose medium containing 10% FBS and 1% double antibody at 37°C and 5% CO2. High-glucose conditions were achieved by adding 40 mM glucose to DMEM. For subsequent experiments, ceAF was added to the medium at various concentrations (0%, 1%, 5%, 10%, and 20%).

[0060] Animal and wound care The experimental protocol was approved by the Animal Care and Ethics Committee of Nanjing Drum Tower Hospital. C57BL / 6 mice (male, 8 weeks old) were obtained from the Model Animal Research Center of Nanjing University and housed in a specific pathogen-free environment with unlimited access to water and food. To create an STZ-induced diabetic animal model, 18 mice in each group received daily intraperitoneal injections of 50 mg / kg streptozotocin (STZ, in sodium citrate buffer) for 5 consecutive days. After 3 weeks, the blood glucose levels of the mice were measured, and mice with blood glucose levels above 16.7 mM were classified as diabetic. To construct the excision wound model, an 8 mm circular biopsy hole was made in the dorsal skin of depilated mice. After modeling, 10% ceAF was applied topically to the wound surface daily, while control mice received the same volume of PBS. Photographs of the wound were taken on days 0, 3, 5, 7, and 11, and the wound area was measured using ImageJ software (National Institutes of Health, USA). Wound tissue samples were collected on days 5 and 10 after wounding for subsequent experiments.

[0061] Histology and immunofluorescence staining Wound edge tissues were fixed, dehydrated, embedded in paraffin, and sectioned at 5 μm thickness. Masson's trichrome (MT), hematoxylin-eosin (H&E), and Sirius red staining were performed according to standard histological procedures. To evaluate macrophage polarization and angiogenesis, the tissues were stained overnight at 4°C using CD206, iNOS, CD31, and α-SMA monoclonal antibodies (1 μg / ml). Next, specific fluorescently labeled secondary antibodies were incubated, followed by DAPI staining.

[0062] RAW264.7 cells were washed sequentially with PBS, fixed with paraformaldehyde (4%), perforated with 0.1% Triton X-100, and blocked with BSA (3%). The cells were then incubated with the corresponding primary and secondary antibodies according to the instructions. All photographs were taken using an Olympus FluoView FV3000 confocal microscope (Tokyo, Japan).

[0063] RNA isolation and RT-qPCR To isolate total RNA, cells and wound edge tissue were treated with Trizol reagent according to the manufacturer's instructions. RT-qPCR was performed using the StepOne RT-qPCR system (Applied Biosystems, USA) with SYBR green dye. After normalization with GAPDH, relative gene levels were determined using the 2-ΔΔCT method. Primer sequences are listed in Table 1.

[0064] [Table 1]

[0065] Western blotting (WB) analysis Protein samples were isolated from lysed skin tissues and cells using RIPA lysis buffer (KeyGEN, China). A BCA assay was performed to determine total protein concentration after centrifugation. Protein samples were separated on a 10% SDS-PAGE gel and transferred to a PDVF membrane (Millipore, USA). After blocking with 5% BSA, the membrane was incubated with the corresponding primary antibody overnight and the secondary antibody for 1 hour. Protein bands were visualized using an ECL substrate kit (Vazyme, China).

[0066] Flow cytometry To confirm the polarization tendency of RAW264.7 macrophages, cells were preincubated with FITC-conjugated anti-mouse F4 / 80 antibody and PE-conjugated anti-mouse CD206 antibody for 30 minutes at 4°C, protected from light. Cell phenotypes were determined using a flow cytometer (FACSCanto II, BD, USA), and data analysis was performed using FlowJo software.

[0067] Cell viability test The viability of RAW264.7 cells was assessed using a CCK-8 assay (Beyotime, China). After starvation for 12 hours, cells were exposed to ceAF at concentrations of 0%, 1%, 5%, 10%, or 20% and then incubated for 24 hours. The cells were washed three times with PBS and then covered with 200 μL of incomplete medium containing the CCK-8 mixture (10 μL) and incubated at 37°C. The absorbance at 450 nm was measured using a microplate reader.

[0068] Enzyme-linked immunosorbent assay (ELISA) RAW264.7 cells were exposed to 10% ceAF for 48 hours, and the cell supernatants were collected for testing. Secreted IL-6, IL-10, TGF-β1, and TNF-α were measured by ELISA kits according to the kit instructions (Elabscience, China).

[0069] statistical analysis Experimental data were analyzed using Graphpad Prism v8.0 software and expressed as mean ± SEM. A parametric test was used for normally distributed data (Shapiro-Wilk test). If the data followed a normal distribution, one-way analysis of variance with a Newman-Keuls post-hoc test was used to compare statistical differences between multiple groups. If the data passed the normality test, a two-tailed Student's t-test was used to compare the two groups. The combined effect of two factors was analyzed by two-way analysis of variance followed by a Tukey post-hoc test. At least three independent measurements were performed, and P < 0.05 was defined as statistically significant.

[0070] Example 1: ceAF attenuates inflammation in lipopolysaccharide-stimulated Raw264.7 cells via the TLR4 / NF-κB signaling pathway To evaluate the regulatory effect of ceAF on lipopolysaccharide (LPS)-induced cellular inflammation, this example used RAW264.7 cells as experimental cells and stimulated them with LPS (100 ng / ml) for 48 hours to induce cellular inflammation.

[0071] First, cell proliferation and viability were measured using various concentrations of ceAF. Based on the results of CCK 8, RAW264.7 proliferation and viability increased with increasing ceAF concentration, peaking at 10% (Figure 1, A). Next, RAW264.7 cells were divided into blank control (NC), LPS-stimulated group (LPS), LPS and ceAF combined intervention group (LPS + ceAF), and ceAF intervention group (ceAF). qPCR results showed that after LPS stimulation, at the transcriptional level, M2 macrophage-related genes (CD 206) decreased, M1 macrophage-related genes (iNOS) increased, and inflammation-related genes (TNF-α, IL-6, IL-1β) increased. These trends were reversed after ceAF intervention (Figure 1, B). Furthermore, Western blotting (WB) experiments were performed at the translational level, and the same trends were observed (Figure 1, C-D).

[0072] Example 2: ceAF induces polarization of RAW264.7 into M2 macrophages in vitro Flow cytometry and cell immunofluorescence confirmed that ceAF induced macrophage polarization into the M2 phase. Flow cytometry labeled macrophages with CD86 and identified M2 macrophages with CD206. Cell immunofluorescence identified M2 macrophages with Arg-1. Results showed that 48 and 72 hours of ceAF intervention effectively increased the proportion of M2 macrophages (Figure 2A-D). Supernatants from RAW264.7 cells were harvested and inflammatory cytokines were measured by ELISA. Results showed that inflammatory cytokines IL-6 and TNF-α were significantly decreased after ceAF intervention, whereas M2-associated secreted factors TGF-β1 and IL-10 were significantly increased after ceAF intervention (Figure 2E).

[0073] Example 3: ceAF promotes wound healing in STZ-induced diabetic mice To evaluate the effect of ceAF on wound healing in streptozotocin (STZ)-induced diabetic mice, 8-week-old male mice were divided into two groups. STZ (50 mg / kg) was intraperitoneally injected for 5 consecutive days. Blood glucose levels were measured one week later, and a diabetic model was considered successful if the blood glucose level exceeded 16.7 mM. A full-thickness wound measuring 8 mm in diameter was created on the back of each mouse using scissors. The control group (DM group) received standard daily medication changes, while the experimental group received daily topical medication changes with 10% ceAF. Photographs of the wound were recorded on days 0, 3, 5, 7, and 11. From day 5 onward, the healing rate in the ceAF group was significantly faster than that in the control group (Figure 3, A-B). On day 11, the body weight and blood glucose levels of the mice were measured, and no significant differences were observed between the two groups (Figure 3, C-D), ruling out the confounding factors of body weight and blood glucose levels.

[0074] Example 4: ceAF improves wound histological parameters in STZ-induced diabetic mice This example further confirmed the effect of ceAF on wound healing in STZ-induced diabetic mice by histopathology. Five days after wound modeling, H&E staining was performed to examine inflammatory cell infiltration in the wound edge tissue, revealing a significant decrease in inflammatory cells in the ceAF group (Figure 4, A-B). Ten days after wound modeling, MT staining was performed to examine collagen deposition in the wound edge tissue, revealing a significant increase in collagen deposition in the ceAF group (Figure 4, C-D). After wound healing in both groups, skin tissue from the healing site was collected and stained with picrosirius red to evaluate the quality of healing (Figure 4, E). The ceAF group contained more type III collagen than the control group, while the ceAF group contained more type I collagen. The ceAF group also contained less scarring, indicating superior healing quality.

[0075] Example 5: ceAF improves wound healing-related indicators in STZ-induced diabetic mice The effect of ceAF on healing-related indicators in STZ-induced diabetic mice was further confirmed by tissue immunofluorescence. Skin wound edge tissue was collected on day 5 and co-stained with F4 / 80 and CD206 to identify the location of M2 macrophages, and with F4 / 80 and iNOS to identify the location of M1 macrophages. In the ceAF group, M2 macrophages were significantly increased at the wound edge (Figure 5, A), while M1 macrophages were significantly decreased (Figure 5, B). Skin wound edge tissue was collected on day 10 and stained for CD31 and α-SMA (Figure 5, C-D). Results indicated a significant increase in neovascularization in the ceAF group.

[0076] Example 6: Regulation of wound-associated inflammatory factors in STZ-induced diabetic mice by ceAF To confirm the effect of ceAF on wound-associated inflammatory factors in STZ-induced diabetic mice in vivo, wound edge tissues were collected 5 days after wound modeling and analyzed by WB and qPCR. The ceAF group showed increased transcription and translation levels of the M2 macrophage genes CD206 and Arg-1 (Figure 6, A, CD), whereas the M1 macrophage marker gene iNOS and the inflammation-related genes TNF-α, IL-6, and IL-1 were decreased (Figure 6, A, E, H).

[0077] These results demonstrate that ceAF ameliorate LPS-induced inflammatory responses in RAW264.7 cells in vitro and promote wound healing in STZ-induced diabetic mice in vivo, through modulation of the TLR4 / NF-κB signaling pathway.

Claims

1. The amniotic fluid is derived from an egg with an embryonic age of 5 to 12 days, preferably an egg with an embryonic age of 6 to 11 days, more preferably an egg with an embryonic age of 7 to 9 days, and even more preferably an egg with an embryonic age of 7 to 8 days, or an egg of a bird other than a chicken whose developmental period corresponds to that of an egg of the embryonic age, or an embryo of a rodent with an embryonic age of 8 to 14 days, or an embryo of a mammal other than a human other than a rodent whose developmental period corresponds to that of a rodent with an embryonic age of 8 to 14 days.

2. The amniotic fluid is derived from an egg with an embryonic age of 5 to 12 days, preferably an egg with an embryonic age of 6 to 11 days, more preferably an egg with an embryonic age of 7 to 9 days, and even more preferably an egg with an embryonic age of 7 to 8 days, or an egg of a bird other than a chicken whose developmental period corresponds to that of an egg of the embryonic age, or an embryo of a rodent with an embryonic age of 8 to 14 days, or an embryo of a mammal other than a human other than a rodent whose developmental period corresponds to that of a rodent with an embryonic age of 8 to 14 days.

3. The amniotic fluid is derived from an egg with an embryonic age of 5 to 12 days, preferably an egg with an embryonic age of 6 to 11 days, more preferably an egg with an embryonic age of 7 to 9 days, and even more preferably an egg with an embryonic age of 7 to 8 days, or an egg of a bird other than a chicken whose developmental period corresponds to that of an egg of the embryonic age, or a rodent embryo with an embryonic age of 8 to 14 days, or an embryo of a mammal other than a human other than a rodent whose developmental period corresponds to that of a rodent with an embryonic age of 8 to 14 days.

4. The amniotic fluid is derived from an egg with an embryonic age of 5 to 12 days, preferably an egg with an embryonic age of 6 to 11 days, more preferably an egg with an embryonic age of 7 to 9 days, and even more preferably an egg with an embryonic age of 7 to 8 days, or an egg of a bird other than a chicken whose developmental period corresponds to that of an egg of the embryonic age, or a rodent embryo with an embryonic age of 8 to 14 days, or an embryo of a mammal other than a human other than a rodent whose developmental period corresponds to that of a rodent with an embryonic age of 8 to 14 days.

5. The amniotic fluid is derived from an egg with an embryonic age of 5 to 12 days, preferably an egg with an embryonic age of 6 to 11 days, more preferably an egg with an embryonic age of 7 to 9 days, and even more preferably an egg with an embryonic age of 7 to 8 days, or an egg of a bird other than a chicken whose developmental period corresponds to that of an egg of the embryonic age, or a rodent embryo with an embryonic age of 8 to 14 days, or an embryo of a mammal other than a rodent whose developmental period corresponds to that of a rodent with an embryonic age of 8 to 14 days. Use of amniotic fluid in the production of a preparation for suppressing the number of M1 macrophage population and increasing the proportion of M2 macrophage types, or a preparation for promoting the transformation of M1 macrophages into M2 macrophages.

6. The application according to any one of claims 1 to 5, characterized in that the medicament or preparation is a cell culture containing the amniotic fluid and / or embryonic stem cells, preferably the medicament or preparation is a pharmaceutical composition containing the amniotic fluid and / or chicken embryonic stem cells and pharmaceutically acceptable excipients.

7. The macrophage-mediated diseases include hypertrophic scars, chronic obstructive pulmonary disease, tumors such as breast cancer and liver cancer, metabolic diseases such as severe obesity, insulin resistance and type 2 diabetes, inflammatory diseases such as acute pancreatitis and arteriosclerosis, cardiovascular diseases such as myocarditis, myocardial infarction and arrhythmia, neurological disorders such as Alzheimer's disease, brain diseases such as cerebral infarction and brain injury, eye diseases such as autoimmune uveitis, retinopathy, keratitis, corneal transplant, Sjogren's syndrome, uveal melanoma and myopia, inflammatory bowel disease, autoimmune hepatitis, asthma, and the like.

2. The method of claim 1, wherein the therapeutic agent is selected from the group consisting of: immunoinflammatory diseases such as rheumatoid arthritis, alcoholic liver disease, colitis, multiple sclerosis, and periodontitis; arthritis such as rheumatoid arthritis, bone erosion, synovitis, and osteoarthritis; acute kidney injury, chronic kidney disease, end-stage renal failure, proliferative glomerulonephritis, membranous nephropathy, diabetic nephropathy, purpura nephritis, ANCA-associated small vessel vasculitis, urinary tract infections, and nephritis such as autosomal dominant polycystic kidney disease; bacterial infections such as sepsis; pregnancy-induced hypertension, diabetes, gestational diabetes, and diabetic nephropathy.

8. 3. The method of claim 2, wherein the M1 macrophage-mediated disease is selected from the group consisting of tumors such as involuting hypertrophic scars, chronic obstructive pulmonary disease, breast cancer, and liver cancer; metabolic diseases such as severe obesity, insulin resistance, and type 2 diabetes; inflammatory diseases such as acute pancreatitis; coronary artery disease such as arteriosclerosis; kidney disease, cardiovascular diseases such as obesity, myocarditis, and myocardial infarction; brain diseases such as cerebral infarction and brain injury; eye diseases such as autoimmune uveitis, retinopathy, keratitis, corneal transplantation, Sjögren's syndrome, and uveal melanoma; immunoinflammatory diseases such as inflammatory bowel disease, autoimmune hepatitis, asthma, alcoholic liver disease, colitis, multiple sclerosis, periodontitis, and osteoarthritis; diabetes, gestational diabetes, and diabetic nephropathy.

9. 4. The method of claim 3, wherein the M2 macrophage-mediated disease is selected from the group consisting of tumors such as proliferative hypertrophic scar, breast cancer, and liver cancer; metabolic diseases such as insulin resistance and type 2 diabetes; inflammatory diseases such as acute pancreatitis; cardiovascular diseases such as myocardial infarction, myocardial failure, arteriosclerosis, coronary artery disease, and myocarditis; ophthalmological diseases such as cerebral infarction, autoimmune uveitis, retinopathy, keratitis, corneal transplant, Sjögren's syndrome, and uveal melanoma; immunoinflammatory diseases such as inflammatory bowel disease, autoimmune hepatitis, asthma, alcoholic liver disease, colitis, multiple sclerosis, periodontitis, and osteoarthritis; diabetes, gestational diabetes, and diabetic nephropathy.

10. The TLR4 / NF-κB signaling pathway mediated diseases include vascular inflammation such as systemic lupus erythematosus, arteriosclerosis, and coronary artery disease, myocarditis such as myocardial ischemic tissue inflammation and myocardial injury, hepatitis such as liver failure, alcoholic liver disease, and inflammatory immune response during alcohol metabolism, fatty liver, pneumonia such as acute lung injury, chronic obstructive pulmonary disease, and silicosis, nephritis such as acute kidney injury and lupus nephritis, inflammatory bowel diseases such as acute enteritis, ulcerative colitis, and radiation proctitis, gastritis such as chronic atrophic gastritis, pneumonia, bronchitis, pharyngitis, sinusitis, and otitis media.

5. The use according to claim 4, characterized in that the therapeutic effect is selected from the group consisting of acute respiratory infections, periodontitis, hyperuricemia, allergic rhinitis, hypersensitivity rhinitis, mastitis, arthritis such as acute gouty arthritis, chronic arthritis, rheumatoid arthritis, inflammation of wound tissue, hypertrophic scars, polycystic ovary syndrome, tumors such as pituitary prolactin adenoma, adrenocorticotropic hormone adenoma, intracranial aneurysms, infectious diseases such as bacterial infections, fungal infections, and viral infections, allergic skin diseases, and allergic diseases such as bronchial asthma, hypersensitivity rhinitis, and allergic purpura.

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