WO2017215602A1 - 多西环素在制备治疗或预防衰老性疾病的药物中的用途 - Google Patents

多西环素在制备治疗或预防衰老性疾病的药物中的用途 Download PDF

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WO2017215602A1
WO2017215602A1 PCT/CN2017/088195 CN2017088195W WO2017215602A1 WO 2017215602 A1 WO2017215602 A1 WO 2017215602A1 CN 2017088195 W CN2017088195 W CN 2017088195W WO 2017215602 A1 WO2017215602 A1 WO 2017215602A1
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doxycycline
cells
use according
aging
caused
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魏炽炬
林浩鹏
王娜
潘峰
郑德锦
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汕头大学
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/65—Tetracyclines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

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  • the present invention relates to a novel use of doxycycline, in particular to the use of doxycycline in the manufacture and/or treatment of a medicament for aging diseases.
  • Doxycycline is an antibiotic of the tetracycline family. It is also known as deoxy oxytetracycline and doxycycline. It is a third-generation semi-synthetic tetracycline broad-spectrum antibiotic. It is in the tetracycline parent ring
  • DOC has the common feature of tetracyclines, which can complex metal ions such as Zn 2+ and Cu 2+ . It mainly acts on the 30S subunit of bacterial ribosome, and interferes with the binding of amino acid tRNA to the site of action on the 30S subunit, blocking the binding of aminoacyl tRNA to the ribosomal mRNA complex, inhibiting protein synthesis and exerting antibacterial action.
  • doxycycline has a variety of other pharmacological effects. For example, studies have shown that doxycycline has anti-inflammatory and anti-oxidative effects. On the other hand, doxycycline can inhibit the activity of matrix metalloproteinases from It acts on tumor cells and affects the process of proliferation, apoptosis, invasion and metastasis of tumor cells. Although there are more and more studies on doxycycline, its biological significance and clinical application have been studied in depth, but so far, there is no research and application of doxycycline in aging diseases.
  • the main object of the present invention is to expand the range of applications of doxycycline, especially its new pharmaceutical use in the prevention and/or treatment of aging diseases, thereby providing new methods and strategies for the treatment and/or prevention of aging diseases. .
  • the technical solution of the present invention is as follows: the use of doxycycline for the preparation of a medicament for treating and/or preventing an aging disease, wherein the doxycycline is administered at a dose equal to or less than 2 ⁇ g/ml; wherein the senescence Diseases include skin aging, neurodegenerative diseases, type 2 diabetes, atherosclerosis, and coronary heart disease.
  • the inventors have found through extensive research that low concentration (different or lower than 2 ⁇ g/ml) of doxycycline can promote the growth of cells (for example, NIH3T3, HeLa cells and INS-1 cells) and decrease the expression of type III collagen. The amount of stress particles in the cells is reduced, and the islet secretion and sensitivity are increased.
  • the concentration of doxycycline is greater than 2 ⁇ g/ml, it has a completely opposite effect in some of the above aspects, resulting in a significant decrease in the overall effect.
  • the aging disease is skin aging.
  • skin aging is prominent, which can be expressed as dry and rough skin, increased wrinkles, deepening, sagging skin and decreased elasticity.
  • Fibroblasts are the most important cellular component in the dermis of the skin and play an important role in the synthesis of secretory fibers and extracellular matrices.
  • the clinical manifestations of skin aging are associated with decreased fibroblast numbers, decreased synthesis or abnormalities.
  • Fibroblasts synthesize and secrete collagen, elastin, collagen fibers, reticular fibers and elastin fibers, as well as synthesize and secrete glycosaminoglycans and glycoproteins. Quality composition.
  • the present inventors have found in the study that doxycycline can significantly promote the growth of fibroblasts when the concentration is as low as 2 ⁇ g/ml.
  • the skin aging is caused by an increase in the expression level of type III collagen.
  • doxycycline can increase the expression of type I collagen, decrease the expression of type III collagen, and achieve skin rejuvenation when the concentration is as low as 2 ⁇ g/ml.
  • the skin aging is caused by an increase in particulate matter secreted by the cells.
  • the particles secreted by the cells are discharged outside the cell, and the surface of the cells is rough.
  • the present inventors have found in the study that when doxycycline is applied at a concentration as low as 2 ⁇ g/ml, the extracellular particulate matter is remarkably reduced, and the cell surface is smoother.
  • the aging disease is a neurodegenerative disease.
  • inorganic sediments inclusion bodies
  • these deposits are formed by abnormal aggregation or amyloidosis of certain proteins and are the main cause of certain neurodegenerative diseases.
  • the neurodegenerative disease is caused by stress granules.
  • Stress granules are sites for storing, repairing, or degrading RNA. If mRNA is not translated properly, these mRNAs are stored in stress granules for repair and can be degraded once they cannot be repaired.
  • An increase in stress granules can lead to various neurodegenerative diseases such as amyotrophic lateral sclerosis and Alzheimer's disease.
  • the neurodegenerative disease is caused by damaging mitochondria.
  • Mitochondria are the metabolic centers of cellular functions and are also the priority targets for the toxic effects of environmental chemical exogenous mitochondria.
  • Mitochondrial damage includes morphological structural damage, mitochondrial DNA mutations, etc.
  • the present inventors have found that normal cells undergo mitochondrial damage and die after treatment with EB (ethidium bromide), and EB-treated cells can still grow in the presence of a suitable concentration of doxycycline. . That is, a suitable concentration of doxycycline protects cells from mitochondrial damage.
  • the aging disease is type 2 diabetes.
  • Diabetes is a disease associated with aging, especially type 2 diabetes. Aging can lead to a severe decline in the ability and sensitivity of islet cells to secrete insulin, and it is difficult to resist external stimuli and eventually lead to loss of function or apoptosis.
  • the type 2 diabetes is caused by apoptosis of islet beta cells.
  • the present inventors have found in the study that doxycycline can significantly promote the proliferation of islet beta cells when the concentration is as low as 2 ⁇ g/ml.
  • the type 2 diabetes is caused by insufficient insulin secretion.
  • the present inventors have found in the study that doxycycline can significantly promote insulin secretion at a concentration as low as 2 ⁇ g/ml.
  • the type 2 diabetes is caused by insulin resistance.
  • Insulin resistance occurs mainly in liver, muscle and adipose tissue.
  • the present inventors have found in the study that doxycycline can significantly promote the growth of liver cells and sensitivity to insulin when the concentration is as low as 2 ⁇ g/ml.
  • the aging disease is caused by high blood fat and high Cholesterol-induced atherosclerosis, coronary heart disease and other cardiovascular diseases.
  • Cardiovascular diseases such as atherosclerosis and coronary heart disease caused by hyperlipidemia and high cholesterol are mainly caused by abnormal fat metabolism pathways, and a large amount of fat and cholesterol are present in the blood, thereby inducing related diseases.
  • the cardiovascular disease is caused by abnormal fat metabolism.
  • the intracellular fat metabolism regulating molecule SREBP sterol regulatory element binding protein
  • SREBP sterol regulatory element binding protein
  • Blocks fat synthesis The inventors found through bioinformatics analysis that doxycycline and SREBP can specifically bind, inhibit SREBP activation, inhibit fat and cholesterol synthesis, reduce intracellular fat droplets, thereby lowering blood lipids and cholesterol levels.
  • the doxycycline is administered at a dose equal to or less than 2 ⁇ g/ml.
  • the doxycycline also comprises various chemically modified various tetracyclines, including: tetracycline, minocycline and the like.
  • doxycycline can effectively promote fibroblast growth, promote type I collagen expression, reduce type III collagen expression, reduce cell division of particulate matter, and achieve cell rejuvenation. Doxycycline also reduces stress granules in cells after EB induction and delays neurodegenerative diseases. On the other hand, doxycycline protects cells from mitochondrial damage, enhances cell viability, and rejuvenates. Moreover, doxycycline can also promote the growth of islet beta cells, and can effectively increase the amount and sensitivity of insulin secretion. Finally, doxycycline specifically binds to the fat metabolism regulatory molecule SREBP, inhibiting the synthesis of fat and cholesterol. Therefore, doxycycline provides new methods and strategies for the treatment and/or prevention of aging diseases.
  • Figure 1 is a microscopic observation of the effect of doxycycline on extracellular particles of NIH3T3 fibroblasts. Figure.
  • Figure 2 is a bar graph showing the effect of doxycycline on the growth of NIH3T3 fibroblasts.
  • Figure 3 is a western blot showing the effect of doxycycline on the expression of type III collagen in NIH3T3 fibroblasts.
  • Figure 4 is a microscopic view showing the effect of doxycycline on HeLa cells and EB-treated HeLa cells.
  • Figure 5 is a bar graph showing the effect of doxycycline on the number of HeLa cells and HeLa cells after EB treatment.
  • Figure 6 is a microscopic view showing the effect of doxycycline on proliferation of mitochondrial-deficient HeLa cells.
  • Figure 7 is a bar graph showing the effect of doxycycline on proliferation of mitochondrial-deficient HeLa cells.
  • Figure 8 is a microscopic view showing that doxycycline promotes the reduction of stress granules in mitochondrial-deficient HeLa cells.
  • Figure 9 is a graph showing immunofluorescence staining of doxycycline promoting the reduction of stress granules in mitochondrial-deficient HeLa cells.
  • Figure 10 is a microscopic view showing that INS-1 islet beta cells were cultured for 24 h under different concentrations of doxycycline.
  • Figure 11 is a microscopic view showing that INS-1 islet beta cells were cultured for 48 h under different concentrations of doxycycline.
  • Figure 12 is a microscopic view showing that INS-1 islet beta cells were cultured for 72 h under different concentrations of doxycycline.
  • Figure 13 is a graph showing flow cytometry of INS-1 islet beta cells cultured at different concentrations of doxycycline for 24h, 48h and 72h.
  • Figure 14 is a bar graph showing the total amount of insulin after incubation of INS-1 islet beta cells for 48 h at different concentrations of doxycycline.
  • Figure 15 is a bar graph showing the high glucose-stimulated insulin secretion of INS-1 islet beta cells cultured for 48 h under different concentrations of doxycycline.
  • Figure 16 is a bar graph showing the effect of doxycycline on insulin secretion sensitivity under high glucose stimulation.
  • Figure 17 is a microscopic view showing the culture of liver cells under doxycycline conditions for 72 hours.
  • Figure 18 is a microscopic view showing the rhodamine-doxycycline complex specifically binding to SREBP and localized around the nucleus and endoplasmic reticulum.
  • Example 1 Doxycycline reduces NIH3T3 fibroblast extracellular particles
  • NIH3T3 cells fibroblasts
  • fibroblasts 1 ⁇ 10 4 NIH3T3 cells (fibroblasts) were seeded per well in a 96-well plate and treated with doxycycline at concentrations of 0, 1 ⁇ g/ml, 2 ⁇ g/ml, 5 ⁇ g/ml, 10 ⁇ g/ml, and 20 ⁇ g/ml, respectively.
  • the cells treated with different concentrations of DOC were photographed under an inverted microscope, and the results are shown in Fig. 1.
  • Fig. 1 1 ⁇ 10 4 NIH3T3 cells (fibroblasts) were seeded per well in a 96-well plate and treated with doxycycline at concentrations of 0, 1 ⁇ g/ml, 2 ⁇ g/ml, 5 ⁇ g/ml, 10 ⁇ g/ml, and 20 ⁇ g/ml, respectively.
  • the cells treated with different concentrations of DOC were photographed under an inverted microscope, and the results are shown in Fig.
  • 0XDOC, 1XDOC, 2XDOC, 5XDOC, 10XDOC, and 20XDOC indicate the concentrations of doxycycline-treated cells of 0, 1 ⁇ g/ml, 2 ⁇ g/ml, 5 ⁇ g/ml, 10 ⁇ g/ml, and 20 ⁇ g/ml, respectively.
  • 10X is a 10:10 objective lens;
  • 20X is a 20:20 objective lens.
  • the amount of particulate matter in the cell culture medium was significantly reduced and the surface of the fibroblasts was smoother after the addition of doxycycline compared with the cells without doxycycline.
  • Example 2 Doxycycline promotes growth of NIH3T3 fiber cells
  • NIH3T3 cells 1 ⁇ 10 4 NIH3T3 cells were inoculated into each well of a 96-well plate, and treated with doxycycline at concentrations of 0, 1 ⁇ g/ml, 2 ⁇ g/ml, 5 ⁇ g/ml, 10 ⁇ g/ml, and 20 ⁇ g/ml, respectively, for 24 hours, 48 hours, and 72 hours. hour.
  • the cells treated with different concentrations of DOC were counted by flow cytometry, and the results are shown in Fig. 2. It can be seen from Fig. 2 that DOC can promote cell growth at 24 h compared with cells without DOC; after 48 h and 72 h, 2 ⁇ g/ml DOC can still promote fibroblast growth. However, as the concentration of DOC increases, it will gradually inhibit the growth of fibroblasts.
  • Example 3 Doxycycline reduces the expression of type III collagen in NIH3T3 fibroblasts
  • NIH3T3 cells 30 ⁇ 10 4 NIH3T3 cells were inoculated into each well of a 6-well plate, treated with doxycycline at a concentration of 0, 2 ⁇ g/ml for 24 hours and 72 hours, respectively, and then total protein was extracted by RAPI lysis to determine the protein concentration after each lane. 50 ⁇ g of total protein was loaded and subjected to western blot, and the results are shown in Fig. 3. As can be seen from Fig. 3, 2 ⁇ g/ml of DOC can significantly reduce the expression level of type III collagen compared to the absence of DOC.
  • Example 4 Effect of doxycycline on HeLa cells and HeLa cells after EB treatment
  • HeLa cells and EB-treated HeLa cells were treated with doxycycline at concentrations of 0, 1 ⁇ g/ml, 2 ⁇ g/ml, 5 ⁇ g/ml, 10 ⁇ g/ml, and 20 ⁇ g/ml, respectively.
  • doxycycline concentrations of 0, 1 ⁇ g/ml, 2 ⁇ g/ml, 5 ⁇ g/ml, 10 ⁇ g/ml, and 20 ⁇ g/ml, respectively.
  • DOC propidium iodide
  • HeLa cells were first treated with EB for 12 days, resulting in the loss of mitochondria in HeLa cells. Then, it was treated with cetamine at a concentration of 0, 1 ⁇ g/ml, 2 ⁇ g/ml, 5 ⁇ g/ml, 10 ⁇ g/ml, and 20 ⁇ g/ml for 4 days.
  • the cells treated with different concentrations of DOC were photographed under an inverted microscope, and the results are shown in Fig. 6.
  • the cells were counted by flow cytometry, respectively, and stained with propidium iodide (PI) to remove dead cells. The results are shown in FIG. As can be seen from Fig. 6 and Fig.
  • Example 6 Effect of doxycycline on mitochondrial-deficient HeLa stress granules.
  • the HeLa cells treated with EB were treated with doxycycline at a concentration of 0, 1 ⁇ g/ml for 4 days, 8 days, and 12 days, respectively. After these three time points, the intracellular stress granules were observed under a microscope, and the cells were simultaneously treated. Immunofluorescence staining (4% paraformaldehyde for 20 minutes, 0.2% Triton for 20 minutes, primary G3BP1 (stress body) for 2 hours, secondary anti-red fluorescence for 1 hour, DAPI for 5 minutes), set Observed by confocal microscopy. The results are shown in Fig. 8 and Fig. 9, and D4, D8, and D2 represent the fourth day, the eighth day, and the twelfth day, respectively.
  • the stress granules i.e., the black particles in the cells in Fig. 8 were added to the cells treated with 1 ⁇ g/ml of DOC compared to the cells to which no DOC was added.
  • the red fluorescence in Figure 9 is significantly reduced.
  • Example 7 Effect of doxycycline on the growth of INS-1 islet Beta cells
  • the 6-well plate of INS-1 cells was collected by trypsinization, resuspended in fresh DMEM, and evenly spread to a 96-well plate at a density of 20%, and added to each other at 0 ⁇ g/ml, 1 ⁇ g. /ml, 2 ⁇ g/ml, 5 ⁇ g/ml, 10 ⁇ g/ml, 20 ⁇ g/ml DOC, 3 replicates per concentration; cultured for 24h, 48h, 72h, respectively, using inverted fluorescence microscope to shoot INS under different DOC concentrations -1 cells 10X, 20X, 40X pictures, each of which takes 3 to 5 fields of view per well, and the results are shown in Figures 10-12.
  • Example 8 Flow cytometry to detect the effect of doxycycline on the growth of INS-1 islet Beta cells
  • the 6-well plate of INS-1 cells was digested with trypsin, resuspended in fresh DMEM, and evenly spread to a 96-well plate at a density of 20%, and added to each other at 0 ⁇ g/ml, 1 ⁇ g/ml, 2 ⁇ g/ MLC, 5 ⁇ g/ml, 10 ⁇ g/ml, DOC at a concentration of 20 ⁇ g/ml, at least 5 duplicate wells per concentration; after 24h, 48h, 72h culture, trypsinize and collect 96-well cells from each group, and flow through
  • the number of INS-1 cells was measured by cytometry, and the results are shown in Fig. 13. As can be seen from Fig.
  • Example 9 ELISA assay for the effect of doxycycline on intracellular insulin in INS-1 cells.
  • the total amount of secreted insulin was measured by ELISA, and the results are shown in Fig. 14.
  • the insulin secretion of INS-1 cells was significantly increased when the DOC concentrations were 1 ⁇ g/ml and 2 ⁇ g/ml compared with the cells without DOC, however, with the increase of DOC concentration, INS The insulin secretion of the -1 cells gradually decreased.
  • Example 10 ELISA assay for glucose-stimulated insulin secretion of INS-1 cells by doxycycline
  • the INS-1 cells were digested with trypsin and collected in 6-well plates, resuspended in fresh DMEM, and evenly spread to a 96-well plate at a density of 30%, respectively, adding 0 ⁇ g/ml, 1 ⁇ g/ml, 2 ⁇ g/ml.
  • the insulin secretion of INS-1 cells increased when the concentration of DOC was 1 ⁇ g/ml and 2 ⁇ g/ml, compared with the cells without DOC. With the increase of DOC concentration, the insulin secretion of INS-1 cells gradually decreased. When the DOC concentration was as high as 20 ⁇ g/ml, insulin secretion was inhibited.
  • Example 11 ELISA assay for insulin secretion at different time points after doxycycline glucose stimulation of INS-1 cells
  • the INS-1 cells were digested with trypsin and collected in 6-well plates, resuspended in fresh DMEM, and evenly spread to a 96-well plate at a density of 30%, and DOC at a concentration of 0 ⁇ g/ml and 1 ⁇ g/ml, respectively. At least 5 replicate wells per concentration; after culture for 48 h, the culture medium was discarded, washed once in KRB buffer, first incubated with glucose-free KRB buffer for 1 h, and then incubated with KRB buffer containing 25 mM glucose for 15 min.
  • Fig. 16 It can be seen from Fig. 16 that under high glucose stimulation, the insulin secretion of INS-1 cells increased at various time points when the concentration of DOC was 1 ⁇ g/ml compared with the cells without DOC. At 15 minutes and 30 minutes after glucose stimulation, the amount of insulin secreted was significantly different.
  • Example 12 Doxycycline promotes growth of LM3 liver cells
  • Example 13 Binding of rhodamine-doxycycline to SREBP and its localization in cells
  • SREBP is associated with the synthesis of fat and cholesterol.
  • the inventors found through bioinformatics analysis that SREBP protein may specifically bind to doxycycline.
  • a rhodamine-doxcycline complex was synthesized by chemical synthesis, and the doxycycline was labeled with red fluorescence, and then passed through
  • Ad293 cells were fixed with 4% paraformaldehyde, washed three times with PBS, and rhodamine-doxcycline complex was added. After incubation for 30 hours, it was washed three times with PBS and observed under a confocal microscope.
  • the cells emit red fluorescence around the nuclear membrane and in the endoplasmic reticulum, indicating that the rhodamine-doxcycline complex specifically binds to substances localized around the nuclear membrane and the endoplasmic reticulum, while SREBP is not activated.
  • SREBP can specifically bind to doxycycline.

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Abstract

本发明提供多西环素在制备治疗和/或预防衰老性疾病的药物中的用途，其中所述多西环素以小于2μg/ml的剂量施用，所述衰老性疾病主要包括皮肤老化、神经退行性疾病、2型糖尿病、线粒体疾病和由高血脂与高胆固醇引发的动脉粥样硬化、冠心病等心血管疾病。

Description

多西环素在制备治疗或预防衰老性疾病的药物中的用途 技术领域
本发明涉及多西环素的新用途，具体地说，涉及多西环素在制备和/或治疗衰老性疾病的药物中的用途。
背景技术
多西环素(Doxycycline，DOC)是四环素家族的一种抗生素，别名脱氧土霉素、强力霉素，属于第三代半合成四环素类广谱抗生素。它是在四环素母环
6α-位上脱氧而得到的一种半合成四环素。DOC具有四环素类药物的共同特征，可以络合金属离子，如Zn2+、Cu2+。主要作用于细菌核蛋白体30S亚基，干扰氨基酸tRNA与30S亚基上的作用位点结合，阻断氨基酰tRNA与核糖体mRNA复合体结合，抑制蛋白质合成而发挥抑菌作用。
Figure PCTCN2017088195-appb-000001
随着对多西环素的深入广泛研究发现，发现多西环素除了很强的抗菌和抗炎作用外，还具有多种其它方面的药理作用。例如，有研究表明多西环素具有抗炎症、抗氧化的作用。另一方面多西环素可以抑制基质金属蛋白酶的活性从 而作用于肿瘤细胞，影响肿瘤细胞的增殖凋亡、侵袭转移等过程。虽然关于多西环素的研究越来越多，其生物学意义和临床应用也获得深入研究，但是，到目前为止，还没有多西环素在衰老性疾病方面的相关研究和应用。
发明内容
本发明的主要目的是扩展多西环素的应用范围，尤其是其在衰老性疾病预防和/或治疗方面的新的制药用途，从而为治疗和/或预防衰老性疾病提供新的方法和策略。
本发明的技术方案如下：多西环素在制备治疗和/或预防衰老性疾病的药物中的用途，其中所述多西环素以等于或小于2μg/ml的剂量施用；其中所述衰老性疾病包括皮肤老化、神经退行性疾病、2型糖尿病、动脉粥样硬化、冠心病。本发明人经过大量的研究发现，低浓度(等于或低于2μg/ml)的多西环素能够促进细胞(例如，NIH3T3，HeLa细胞和INS-1细胞)的生长，降低III型胶原蛋白表达量，减少细胞中的应激性颗粒，增加胰岛分泌量和敏感性等作用。而一旦多西环素的作用浓度大于2μg/ml，其在上述某些方面具有完全相反的作用，导致总体作用效果大幅下降。
作为对上述技术方案的进一步限定，其中所述衰老性疾病为皮肤老化。随着年龄增长，皮肤老化突显，可表现为皮肤干燥粗糙，皱纹增多、加深，皮肤松弛，弹性下降。
作为对上述技术方案的进一步限定，其中所述皮肤老化由成纤维细胞数量减少而引起。成纤维细胞是皮肤真皮中最主要的细胞成分，在合成分泌纤维和细胞外基质中扮演着重要的角色。皮肤老化的临床表现都与成纤维细胞数量减少、合成功能下降或异常有关。成纤维细胞既合成和分泌胶原蛋白、弹性蛋白，生成胶原纤维、网状纤维和弹性纤维，也合成和分泌糖胺多糖和糖蛋白质等基 质成分。本发明人在研究中发现，多西环素在低至2μg/ml的浓度作用时，可以明显促进成纤维细胞的生长。
作为对上述技术方案的进一步限定，其中所述皮肤老化由III型胶原蛋白表达量提高而引起。研究发现在皮肤成纤维细胞的衰老过程中胶原蛋白的表达分泌会发生明显改变。胶原蛋白随着年龄的增加而流失、结构发生变化，组成比例和代谢平衡都有变化。皮肤结构的松弛、皱纹的产生都与胶原蛋白息息相关。在婴儿和青年人皮肤中，I型胶原蛋白含量约占70％，III型胶原蛋白占30％。在衰老过程中，成纤维细胞合成III型胶原蛋白增加，I型胶原蛋白减少。本发明人在研究中发现，多西环素在低至2μg/ml的浓度作用时，可以提高I型胶原蛋白表达量，降低III型胶原蛋白表达量，实现皮肤年轻化。
作为对上述技术方案的进一步限定，其中所述皮肤老化由细胞分泌的颗粒物增加而引起。细胞分泌的颗粒物会排到细胞外，而且细胞表面粗糙。本发明人在研究中发现，多西环素在低至2μg/ml的浓度作用时，细胞外的颗粒物质明显减少，细胞表面更光滑。
作为对上述技术方案的进一步限定，其中所述衰老性疾病为神经退行性疾病。近年来采用组织化学方法在人脑中陆续发现不溶性沉积物(包涵体)，进一步研究发现，这些沉积物是由于某些蛋白质异常聚集或淀粉样化形成的，是某些神经退行性疾病的主要致病原因。
作为对上述技术方案的进一步限定，其中所述神经退行性疾病由应激性颗粒而引起。应激性颗粒是用于储存、修复或者降解RNA的场所，如果mRNA不能正常翻译，这些mRNA会被储存在应激性颗粒中，进行修复，一旦无法修复，会被降解。应激性颗粒的增加，会导致各种神经退行性疾病，例如，肌萎缩性脊髓侧索硬化症、阿兹海默症等。本发明人在研究中发现，多西环素在低 至2μg/ml的浓度作用时，可以明显减少细胞内的应激性颗粒。
作为对上述技术方案的进一步限定，其中所述神经退行性疾病由损伤性线粒体而引起。线粒体是细胞功能代谢中心，同时也是环境化学外源物毒害作用的优先靶标，线粒体损伤包括形态结构破坏、线粒体DNA突变等，线粒体与细胞衰老之间有明密切的关系。本发明人在研究中发现，正常细胞经过EB(溴化乙锭)处理后，会发生线粒体损伤而死亡，而在适合浓度的多西环素存在的情况下，经过EB处理的细胞依然可以生长。也就是说，适合浓度的多西环素可以保护细胞免受线粒体损伤的影响。
作为对上述技术方案的进一步限定，其中所述衰老性疾病为2型糖尿病。糖尿病是一种与衰老相关的疾病，特别是2型糖尿病，衰老会导致胰岛细胞分泌胰岛素能力和敏感性严重下降，而且难以抵抗外界刺激压力，最终导致功能缺失或者凋亡。
作为对上述技术方案的进一步限定，其中所述2型糖尿病为胰岛beta细胞凋亡引起。本发明人在研究中发现，多西环素在低至2μg/ml的浓度作用时，可以明显促进胰岛beta细胞的增殖。
作为对上述技术方案的进一步限定，其中所述2型糖尿病为胰岛素分泌不足引起。本发明人在研究中发现，多西环素在低至2μg/ml的浓度作用时，可以明显促进胰岛素的分泌。
作为对上述技术方案的进一步限定，其中所述2型糖尿病为胰岛素阻抗引起。胰岛素阻抗主要发生在肝脏、肌肉和脂肪组织。本发明人在研究中发现，多西环素在低至2μg/ml的浓度作用时，可以明显促进肝脏细胞的生长及对胰岛素的敏感性。
作为对上述技术方案的进一步限定，其中所述衰老性疾病为由高血脂与高 胆固醇引发的动脉粥样硬化、冠心病等心血管疾病。由高血脂与高胆固醇引发的动脉粥样硬化、冠心病等心血管疾病主要是由于脂肪代谢通路异常，大量脂肪与胆固醇存在于血液中，从而诱发相关疾病。
作为对上述技术方案的进一步限定，其中所述心血管疾病为脂肪代谢异常引起。在胆固醇水平不足的情况下，细胞内的脂肪代谢调节分子SREBP(固醇调节元件结合蛋白)会被激活并与细胞DNA结合，促进脂肪合成；在胆固醇水平过高的情况下，SREBP受到抑制，阻碍脂肪合成。本发明人通过生物信息学分析发现，多西环素与SREBP可发生特异性结合，抑制SREBP激活，阻碍脂肪和胆固醇合成，减少细胞内的脂肪滴，从而降低血脂和胆固醇水平。
作为对上述技术方案的进一步限定，其中所述多西环素以等于或小于2μg/ml的剂量施用。
作为对上述技术方案的进一步限定，其中所述多西环素也包含化学修饰的各种四环素，包括：四环素、米诺霉素等。
本发明人发现，多西环素能有效促进成纤维细胞生长，促进I型胶原蛋白表达，降低III型胶原蛋白的表达，减少细胞分裂的颗粒物质，实现细胞的年轻化。多西环素还能减少细胞在EB诱导后的应激性颗粒，延缓神经退行性疾病。另外一方面，多西环素可以保护细胞免受线粒体损伤的影响，实现细胞的活力增强，并实现年轻化。而且，多西环素能还促进胰岛beta细胞的生长，而且能有效提高胰岛素的分泌量和敏感性。最后，多西环素能与脂肪代谢调节分子SREBP特异性结合，抑制脂肪和胆固醇的合成。因此，多西环素为治疗和/或预防衰老性疾病提供新的方法和策略。
附图说明
图1为显示多西环素对NIH3T3成纤维细胞外颗粒物的影响的显微镜观察 图。
图2为显示多西环素对NIH3T3成纤维细胞生长的影响的柱状图。
图3为显示多西环素对NIH3T3成纤维细胞III型胶原蛋白表达量的影响的western blot图。
图4为显示多西环素对HeLa细胞和EB处理后的HeLa细胞的影响的显微镜观察图。
图5为显示多西环素对HeLa细胞和EB处理后的HeLa细胞数量的影响的柱状图。
图6为显示多西环素对线粒体缺陷型HeLa细胞增殖的影响的显微镜观察图。
图7为显示多西环素对线粒体缺陷型HeLa细胞增殖的影响的柱状图。
图8为显示多西环素促进线粒体缺陷型HeLa细胞内应激性颗粒减少的显微镜观察图。
图9为显示多西环素促进线粒体缺陷型HeLa细胞内应激性颗粒减少的免疫荧光染色图。
图10为显示INS-1胰岛beta细胞在不同浓度多西环素条件下培养24h的显微镜观察图。
图11为显示INS-1胰岛beta细胞在不同浓度多西环素条件下培养48h的显微镜观察图。
图12为显示INS-1胰岛beta细胞在不同浓度多西环素条件下培养72h的显微镜观察图。
图13为显示INS-1胰岛beta细胞在不同浓度多西环素条件下培养24h、48h和72h的流式细胞计数图。
图14为显示INS-1胰岛beta细胞在不同浓度多西环素条件下培养48h后胰岛素总量的柱状图。
图15为显示INS-1胰岛beta细胞在不同浓度多西环素条件下培养48h后高糖刺激胰岛素分泌量的柱状图。
图16为显示多西环素对高糖刺激下的胰岛素分泌敏感性影响的柱状图。
图17为显示肝脏细胞在多西环素条件下培养72h的显微镜观察图。
图18为显示罗丹明-多西环素复合物与SREBP特异性结合，并定位在细胞核周围与内质网的微镜观察图。
具体实施方式
实施例1：多西环素减少NIH3T3成纤维细胞外颗粒物
在96孔板每孔接种1x104个NIH3T3细胞(成纤维细胞)，分别用0、1μg/ml、2μg/ml、5μg/ml、10μg/ml、20μg/ml浓度的多西环素处理。在倒置显微镜下分别对不同浓度DOC处理后的细胞进行拍照，结果如图1所示。在图1中，0XDOC、1XDOC、2XDOC、5XDOC、10XDOC、20XDOC分别表示多西环素处理细胞的浓度为0、1μg/ml、2μg/ml、5μg/ml、10μg/ml、20μg/ml。10X为10:10倍物镜；20X为20:20倍物镜。由图1可以看出，与未加多西环素的细胞相比，加入多西环素后，细胞培养液中的颗粒物质明显减少，成纤维细胞表面更为光滑。
实施例2：多西环素促进成NIH3T3纤维细胞生长
在96孔板每孔接种1x104的NIH3T3细胞，分别用0、1μg/ml、2μg/ml、5μg/ml、10μg/ml、20μg/ml浓度的多西环素处理24小时、48小时、72小时。三个时间点后，分别用流式细胞仪对不同浓度DOC处理后的细胞进行计数，结果如图2 所示。由图2可以看出，与没有加入DOC的细胞相比，在24h时，各个浓度的DOC都可以促进细胞生长；到了48h和72h后，2μg/ml的DOC依然可以促进成纤维细胞的生长，然而随着DOC浓度的增加，反而会逐渐抑制成纤维细胞的生长。
实施例3：多西环素减少NIH3T3成纤维细胞III型胶原蛋白的表达量
在6孔板每孔接种30x104的NIH3T3细胞，用0、2μg/ml浓度的多西环素分别处理24小时和72小时，然后用RAPI裂解法提取总蛋白，测定蛋白浓度后，每条泳道上样50μg总蛋白，进行western blot，结果如图3所示。由图3可以看出，与没有加入DOC相比，2μg/ml的DOC可以明显减低III型胶原蛋白的表达量。
实施例4：多西环素对HeLa细胞和EB处理后HeLa细胞的影响
将HeLa细胞和加入EB处理的HeLa细胞(加入EB后，细胞处于应激状态)分别用0、1μg/ml、2μg/ml、5μg/ml、10μg/ml、20μg/ml浓度的多西环素处理4天，在倒置显微镜下分别对不同浓度DOC处理后的细胞进行拍照，结果如图4所示。同时，分别用流式细胞仪对细胞进行计数，计数前经碘化丙啶(PI)染色，除去死亡细胞，结果如图5所示。由图4和图5(A)可以看出，对于未经EB处理的HeLa细胞而言，与没有加入DOC的细胞相比，1μg/ml和2μg/ml的DOC可以促进细胞的生长，然而随着DOC浓度的增加，反而会逐渐抑制细胞的生长；由图4和图5(B),可以看出对于经EB处理的HeLa细胞而言，未加入DOC时，细胞生长状态明显下降，加入DOC可以促进细胞的生长，尤其是DOC的浓度为1μg/ml和2μg/ml时，促进作用非常明显，然而随着DOC 浓度的增加，促进作用逐渐减弱，但依然存在促进作用。可以看出，合适浓度的多西环素可以保护细胞免受线粒体损伤的影响，并促进细胞的生长。
实施例5：多西环素对线粒体缺陷型细胞的影响
先用EB处理HeLa细胞12天，使得HeLa细胞的线粒体发生缺失。然后分别用0、1μg/ml、2μg/ml、5μg/ml、10μg/ml、20μg/ml浓度的西环素处理4天。在倒置显微镜下分别对不同浓度DOC处理后的细胞进行拍照，结果如图6所示。同时，分别用流式细胞仪对细胞进行计数，计数前经碘化丙啶(PI)染色，除去死亡细胞，结果如图7所示。由图6和图7可以看出，对于经EB处理的线粒体已发生缺失的HeLa细胞，与没有加入DOC的细胞相比，随着DOC浓度的增加，细胞量也逐渐增加，也就是线粒体缺失的条件下，低浓度DOC不足以保护细胞，而高浓度DOC才可以保护细胞，表明DOC不但可以通过调节线粒体而促进细胞生长的，而且可能在线粒体缺失的条件下，高浓度DOC可以通过其他通路保护细胞，促进细胞生长。
实施例6：多西环素对线粒体缺陷型HeLa应激性颗粒的影响。
用0、1μg/ml浓度的多西环素分别处理EB处理后HeLa细胞4天、8天和12天，在这三个时间点后用显微镜观察细胞内的应激性颗粒，同时对细胞进行免疫荧光染色(4％多聚甲醛固定20分钟，0.2％曲拉通处理20分钟，一抗G3BP1(应激小体)孵育2小时，二抗红色荧光孵育1小时，DAPI染色5分钟)，置于共聚焦显微镜观察。结果如图8和图9所示，D4、D8、D2分别代表第4天、第8天、第12天。由图8和图9可以看出，与没有加入DOC的细胞相比，加入1μg/ml DOC处理的细胞中的应激性颗粒(即图8中细胞内的黑色颗粒物， 图9中的红色荧光)明显减少。
实施例7：多西环素对INS-1胰岛Beta细胞生长的影响
用胰酶消化并收集6孔板的INS-1细胞(胰岛瘤细胞)，用新鲜的DMEM重悬后，将其以20％的密度均匀铺到96孔板，并分别加入0μg/ml,1μg/ml,2μg/ml,5μg/ml,10μg/ml,20μg/ml浓度的DOC，每个浓度3个复孔；分别培养24h，48h，72h后，用倒置荧光显微镜拍摄不同DOC浓度条件下INS-1细胞10X、20X、40X的图片，每个浓度每个孔分别取3～5个视野，结果如图10～12所示。由图10～12可以看出，与没有加入DOC的细胞相比，当DOC浓度为1μg/ml和2μg/ml时，可以明显促进INS-1细胞的生长，然而，随着DOC浓度的增加，这种促进作用明显逐渐减弱。
实施例8：细胞流式术检测多西环素对INS-1胰岛Beta细胞生长的影响
用胰酶消化并收集6孔板的INS-1细胞，用新鲜的DMEM重悬后，将其以20％的密度均匀铺到96孔板，并分别加入0μg/ml,1μg/ml,2μg/ml,5μg/ml,10μg/ml,20μg/ml浓度的DOC，每个浓度至少5个复孔；分别培养24h、48h、72h后，用胰酶消化并收集各组96孔细胞，通过流式细胞术检测INS-1细胞数，结果图13所示。由图13可以看出，与没有加入DOC的细胞相比，当DOC浓度为1μg/ml和2μg/ml时，可以明显促进INS-1细胞的生长，然而，随着DOC浓度的增加，这种促进作用明显逐渐减弱，该结果与实施例7的结果一致。
实施例9：ELISA法检测多西环素对INS-1细胞胞内胰岛素的影响。
用胰酶消化并收集6孔板的INS-1细胞，用新鲜的DMEM重悬后，将其以 30％的密度均匀铺到96孔板，并分别加入0μg/ml,1μg/ml,2μg/ml,5μg/ml,10μg/ml,20μg/ml浓度的DOC浓度的DOC，每个浓度至少5个复孔；培养48h后，弃培养液，PBS洗2次，胰酶消化并收集各组细胞沉淀，1.5％盐酸乙醇孵育20分钟，-80℃/4℃反复冻融3次，离心取上清，用ELISA检测分泌的胰岛素的总量，结果如图14所示。由图14可以看出，与没有加入DOC的细胞相比，当DOC浓度为1μg/ml和2μg/ml时，INS-1细胞的胰岛素分泌量明显增加，然而，随着DOC浓度的增加，INS-1细胞的胰岛素分泌量逐渐降低。
实施例10：ELISA法检测多西环素对INS-1细胞的葡萄糖刺激的胰岛素分泌量
用胰酶消化并收集6孔板的INS-1细胞，用新鲜的DMEM重悬后，将其以30％的密度均匀铺到96孔板，分别加入0μg/ml,1μg/ml,2μg/ml,5μg/ml,10μg/ml,20μg/ml浓度的DOC，每个浓度至少5个复孔；培养48h后，弃培养液，KRB缓冲液洗1次，先用不含葡萄糖的KRB缓冲液孵育1h，再用含25mM葡萄糖的KRB缓冲液孵育8小时，收集上清，PBS洗涤2次，用RIPA裂解细胞，收集并离心，取上清进行BCA蛋白测定。结果如图15所示。由图15可以看出，在高浓度的葡萄糖刺激下，与没有加入DOC的细胞相比，当DOC浓度为1μg/ml和2μg/ml时，INS-1细胞的胰岛素分泌量增加，然而，随着DOC浓度的增加，INS-1细胞的胰岛素分泌量逐渐降低，当DOC浓度高至20μg/ml时，胰岛素的分泌被抑制。
实施例11：ELISA法检测多西环素对INS-1细胞的葡萄糖刺激后不同时间点的胰岛素分泌量
用胰酶消化并收集6孔板的INS-1细胞，用新鲜的DMEM重悬后，将其以30％的密度均匀铺到96孔板，分别加入0μg/ml和1μg/ml浓度的DOC，每个浓度至少5个复孔；培养48h后，弃培养液，KRB缓冲液洗1次，先用不含葡萄糖的KRB缓冲液孵育1h，再用含25mM葡萄糖的KRB缓冲液孵育后的15min、30min、1h、2h和4h 5个时间点，收集上清，PBS洗涤2次，用RIPA裂解细胞，收集并离心，取上清进行BCA蛋白测定，结果如图16所示。由图16可以看出，在高浓度的葡萄糖刺激下，与没有加入DOC的细胞相比，当DOC浓度为1μg/ml时，在各个时间点INS-1细胞的胰岛素分泌量均有增加，尤其在葡萄糖刺激后15分钟和30分钟时，胰岛素的分泌量相差显著。
实施例12：多西环素促进成LM3肝脏细胞生长
在96孔板每孔接种1x104个LM3肝脏细胞，分别用0和1μg/ml浓度的多西环素处理。在倒置显微镜下分别对不同浓度DOC处理后的细胞进行拍照，结果如图17所示。在图17中，0XDOC、1XDOC、分别表示多西环素处理细胞的浓度为0、1μg/ml。10X为10:10倍物镜。由图17可以看出，与未加多西环素的细胞相比，加入多西环素后，细胞培养液中的颗粒物质明显减少，LM3肝脏细胞表面更为光滑，细胞生长加快，说明多西环素有利于降低胰岛素阻抗，因为肝脏细胞的胰岛素阻抗是产生2型糖尿病的一个重要原因。
实施例13：罗丹明-多西环素与SREBP的结合及其在细胞中的定位
SREBP与脂肪和胆固醇的合成相关，本发明人经过生物信息学分析发现，SREBP蛋白可能与多西环素特异性结合。为了验证此推断，利用化学合成方法，合成了罗丹明-多西环素复合物，使多西环素化被标记上红色荧光，然后通过免 疫化学方法，对Ad293细胞用4％多聚甲醛固定，PBS洗三次，加入罗丹明-多西环素复合物，孵育30小时后用PBS洗三次，置于共聚焦显微镜下观察。可以观察得到，细胞的核膜周围和内质网处发出红色荧光，表明罗丹明-多西环素复合物与定位在核膜周围和内质网的物质特异性结合，而SREBP在没有被激活的情况下，是定位在核膜周围和内质网处，因此，SREBP可与多西环素特异性结合。

Claims (15)

  1. 多西环素在制备治疗和/或预防衰老性疾病的药物中的用途，其中所述多西环素以等于或小于2μg/ml的剂量施用；
    其中所述衰老性疾病包括皮肤老化、神经退行性疾病、2型糖尿病、动脉粥样硬化、冠心病。
  2. 如权利要求1所述的用途，其中所述衰老性疾病为皮肤老化。
  3. 如权利要求2所述的用途，其中所述皮肤老化由成纤维细胞数量减少而引起。
  4. 如权利要求2所述的用途，其中所述皮肤老化由I型胶原蛋白表达量下降而III型胶原蛋白表达量提高而引起。
  5. 如权利要求2所述的用途，其中所述皮肤老化由细胞分泌的颗粒物增加而引起。
  6. 如权利要求1所述的用途，其中所述衰老性疾病为神经退行性疾病。
  7. 如权利要求6所述的用途，其中所述神经退行性疾病由应激性颗粒引起。
  8. 如权利要求6所述的用途，其中所述神经退行性疾病由损伤性线粒体而引起。
  9. 如权利要求1所述的用途，其中所述衰老性疾病为2型糖尿病。
  10. 如权利要求9所述的用途，其中所述2型糖尿病由胰岛Beta细胞增殖障碍引起。
  11. 如权利要求9所述的用途，其中所述2型糖尿病由胰岛素释放不足引起。
  12. 如权利要求9所述的用途，其中所述2型糖尿病由胰岛素阻抗引起。
  13. 如权利要求1所述的用途，其中所述衰老性疾病为动脉粥样硬化和/或冠心病。
  14. 如权利要求13所述的用途，其中所述动脉粥样硬化和/或冠心病由脂肪代谢异常引起。
  15. 如权利要求1所述的用途，其中所述多西环素包含化学修饰的四环素和米诺霉素。
PCT/CN2017/088195 2016-06-16 2017-06-14 多西环素在制备治疗或预防衰老性疾病的药物中的用途 WO2017215602A1 (zh)

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