CN104510736A - Compound for activating AMPK and application thereof - Google Patents
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Abstract
The invention discloses a compound for activating AMPK. The compound is adenine and/or pharmaceutically-acceptable salts thereof. The invention further discloses the application of the compound. The compound is used to activate AMPK (AMP-kinase) and prevent/treat physiological discomfort or diseases. Thus the compound can be applied to prevention and treatment on physiological discomfort or diseases that can be relieved by AMPK for mammals.
Description
Technical field
The present invention is about adenine, and this compound is applicable to activation AMPK (AMP-activated protein kinase), and uses this compound in prevention or treat physiological situation or disease.
Background technology
AMPK is clearly the induction apparatus of cellular energy and the response person of energy requirement.AMPK is that different triplet is made up of catalytic α sub-cell body, modulability β, γ sub-cell body, and all sub-cell bodies are in eukaryote tool height retention.AMPK activation is as LKB1, calcium ion/take the plain protein phosphokinase (Ca relied on of calcium by its upstream kinases
2+/ Calmodulin dependent kinase) and the 172nd Soviet Union's amino acid residue of TAK1 phosphorylation α sub-cell body tool retention, cause high AMP/ATP ratio also to activate AMPK by physiology or pathology pressure.Can promote that catabolism path is carried out and suppresses anabolism after AMPK activation, consume by minimizing ATP and promote ATP to generate and then recover cellular energy balance.
As an energy metabolism balance moderator, AMPK is considered to metabolic syndrome, comprises the medicine target that Second-Type diabetes, cardiovascular disease, fatty liver etc. are tool potentiality.Many metabolic syndromes are all relevant with insulin resistant.Insulin resistant is a pathological state, and cannot respond insulin at this situation cell, therefore too much in blood glucose cannot remove to skeletal muscle or fatty tissue.In muscle cell, AMPK activation, in non-insulin-dependent mode, increases glucose transport protein (GLUT4) performance amount by transcriptional control, and induces GLUT4 to be transferred on cell membrane to cause cellular uptake glucose speed to increase.AMPK activation also suppresses fatty acid and cholesterol biosynthesis by suppression acetyl-CoA carboxylase (acetyl-CoA carboxylase) and Hydroxymethylglutaryl list acyl coenzyme A reductase (HMG-CoA reductase) respectively.In addition AMPK activation causes the suppression of several transcription factor, comprises SREBP-1c, ChREBP and HNF-4a, the protein performance of ferment and decline adjustment fatty acid synthesis and gluconeogenesis effect are correlated with.The above-mentioned research mentioned finds, all supports that AMPK is the treatment target for metabolic syndrome especially diabetes.
Except energy metabolism balance regulate except, AMPK also participates in the adjustment of several cell mechanism, comprise inflammatory response, Growth of Cells, cell carving die, autophagy, aging and differentiation.Many research display AMPK are inflammatory response suppression person.AMPK activation suppresses inflammatory response by the message transmission of T suppression cell nuclear factor (NF-κ B).The message transmission of nuclear factor is activation innate immunity and acquired immunity predominating path, when meeting after AMPK activation is by stimulating the transcriptional activity of SIRT1, Forkhead box O (FoxO) or peroxisome proliferator-activated receptor co-activator 1 α (PGC1 α) T suppression cell nuclear factor to reach the effect suppressing inflammatory response.In addition several research department also proves that AMPK activation suppresses the protein performance of Transitional cell carcinomas (cyclooxygenase-2, COX-2).Transitional cell carcinomas is an inductivity ferment, can be regulated and controled by inflammatory cytohormone and somatomedin, thus its function causes inflammatory response and pain for arachidonic acid is changed into prostaglandin, therefore suppresses the activity of Cycloxygenase or performance to be proved the effect of tool anti-inflammatory.
Several AMPK activator is proved tool anti-inflammatory function in biological experiment in vivo.For example, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) has been proved the acute and recurrent colitis can alleviated caused by trinitro-benzene-sulfonic acid or dextran sodium sulfate in mouse model, AICAR treatment significantly reduces losing weight and slowing down inflammatory response of disease mice.In addition AICAR has obvious therapeutic effect for multiple sclerosis zootype (EAE), also reduces with the order of severity of lipopolysaccharide inducing mouse injury of lung.
The imbalance of cell message bang path may cause abnormal growth of cells, finally causes cancer.Mammal Rapamycin albumen (mTOR) is an amino acid/Soviet Union's amine acid kinase, the growth of its regulating cell and autophagocytosis.The activity imbalance of mammal Rapamycin albumen message bang path is found in many various cancers, and therefore mammal Rapamycin protein inhibitor is considered to the potential drugs for the treatment of of cancer.Large quantity research confirms that AMPK phosphorylation tuberous sclerosis complex 2 (TSC2) and Raptor reaches and suppresses mammal Rapamycin albumen path.Various AMPK activator comprises AICAR, metformin, phenformin and has been proved suppression mammal Rapamycin albumen message path, and anticancer growth.In addition AMPK activation is by suppressing mammal Rapamycin protein complexes-1 inducing self-body phagocytosis.Because AMPK suppresses the upper 757th amino acid phosphorylation minimizing of mammal Rapamycin protein complexes-1, Ulk1, thereafter the 317th and 777 amino acids by AMPK phosphorylation, Ulk1 by after AMPK phosphorylation with startup autophagocytosis.
In sum, AMPK is considered to many human diseasess or pathological condition, and comprising inflammatory diseases, wound healing, nerve degeneration, cancer, oxidative pressure and cardiovascular disease is one well treat target.In fact, AMPK activator has been applied to the clinical trial of at least 24 class diseases, comprising: antibacterial and fungal disease, behavior and mental disorder, blood and lymphatic disease, cancer, tumor, digestive system disease, otolaryngologic disease, ophthalmic, body of gland and He Ermeng relevant disease, cardiovascular disease, disease of immune system, face and odontopathy, muscle, skeleton, cartilage disease, nervous system disease, nutrition and metabolic disease, respiratory tract disease, skin and connective tissue disease, wound healing etc.
Summary of the invention
The invention provides a kind of novel AMPK activator-adenine and use this compound in prevention or disease therapy.
The invention provides a kind of compound for activating AMPK, it is adenine and/or its pharmaceutically acceptable salt.
The invention provides above-claimed cpd treats the medicine of disease or the physiological situation that can be improved by AMPK activator application as preparation.
The invention provides above-claimed cpd treats the medicine of inflammatory physiological situation or disease application as preparation.
The invention provides the application of above-claimed cpd as preparation prevention or treatment pre-diabetes, Second-Type diabetes, metabolic syndrome a kind of or physiological situation of its combination or the medicine of disease.
The invention provides the application of above-claimed cpd as preparation prevention or treatment pre-diabetes, Second-Type diabetes, metabolic syndrome a kind of or physiological situation of its combination or the medicine of disease.
The invention provides above-claimed cpd as the application preparing the medicine preventing or treat Alzheimer disease.
The invention provides above-claimed cpd treats the medicine of disease or the physiological situation that can be improved by autophagocytosis application as preparation.
The invention provides above-claimed cpd as the application of medicine preparing wound healing process and suppress cicatrization.
The invention provides above-claimed cpd strengthens the medicine of wound healing application as preparation.
The invention provides the application of the medicine of the cell that above-claimed cpd injures as protection in preparation mammal and treatment receptor 1 activity chalcogen.
The invention provides above-claimed cpd as the application preparing prevention or Therapeutic cancer medicine.
According to embodiments of the invention, provide a novel AMPK activator-adenine, in intracellular activation AMPK, therefore in mammal prevention or can treat the physiological situation or disease that can be improved by AMPK.
According to embodiments of the invention, one is provided to fall hypoglycemic method by activation AMPK, thus prevention or disease therapy comprise: metabolic syndrome, pre-diabetes, Second-Type diabetes, insulin resistant, wherein need the adenine of this mammal effective amounts for the treatment of and/or pharmaceutically acceptable salt.
According to embodiments of the invention, provide one by activating the method for AMPK anti-inflammatory, thus prevent or treat inflammatory situation or disease, wherein need the adenine of this mammal effective amounts for the treatment of and/or pharmaceutically acceptable salt.
According to embodiments of the invention, provide one to suppress the method for Fibroblast Growth by activation AMPK, thus prevention of scar tissue generate during wound healing.
According to embodiments of the invention, provide the method for a reinforcement wound healing, wherein need the adenine of this mammal effective amounts for the treatment of and/or pharmaceutically acceptable salt.
According to embodiments of the invention; the one suppression method that reactive chalcogen (ROS) generates is provided; thus protection or treat mammiferous cell and injure away from reactive chalcogen, the adenine of the mammal effective amounts wherein needing this to treat and/or pharmaceutically acceptable salt.
According to embodiments of the invention, provide the method for an anticancer growth, thus prevention or Therapeutic cancer, wherein need the adenine of this mammal effective amounts for the treatment of and/or pharmaceutically acceptable salt.
The invention relates to adenine, this compound is applicable to activate AMPK, and use adenine in prevention or treat physiological situation or disease, comprising: pre-diabetes, insulin resistant, Second-Type diabetes, metabolic syndrome, obesity, inflammation, wound healing, Alzheimer disease, cancer, oxidative pressure and cardiovascular disease.
The present invention finds, adenine is the AMPK activator of a novelty, and has various biological function.In recent years, AMPK activation has been proved the Prevention and Curation contributing to disease, as pre-diabetes, insulin resistant, Second-Type diabetes, metabolic syndrome, obesity, inflammation, wound healing, Alzheimer disease, cancer, oxidative pressure, cardiovascular disease and promotion wound healing.It is considered herein that, this effect is attributable to AMPK activation institute and causes but be not limited to reduce Transitional cell carcinomas performance amount, the production of suppression answering property chalcogen (ROS) and the increase of glucose uptake activity.
Expection indication
Based on achievement in research of the present invention (see embodiment), adenine is by activating AMPK as the therapeutic agent being used in various physiological situation or disease.Example guidance and the evidence of expection indication are below provided.
Adenine is in the treatment of hyperglycemia, pre-diabetes, insulin resistant, Second-Type diabetes
Existing report recently points out that AMPK activator comprises metformin, and A769662, AICAR reduce plasma glucose concentration in diabetes or obesity mice pattern.In the present invention, the adenine of 1 μM ~ 600 μMs significantly increases the glucose uptake (table 2) of muscle cell C2C12.Further feed mice as Second-Type diabetes animal model assessment adenine to plasma glucose concentration regulating effect using high fat diet.Feed mice with the high fat diet of control group to compare, give adenine and significantly reduce the plasma glucose more than 30% that high fat diet feeds mice, and reduce lose weight (embodiment 3) of blood plasma triglyceride more than 35%, more than 15%.Term used herein " hyperglycemia " refers to a physiological situation, it is characterized by blood glucose higher than 126 milligrams/deciliter.Term used herein " pre-diabetes " refers to a physiological situation, it is characterized by fasting glucose higher than 100 milligrams/deciliter but lower than 140 milligrams/deciliter.Term used herein " insulin resistant " refers to a physiological situation, and wherein whole body or tissue comprise liver, skeletal muscle, fatty tissue cannot to insulin response.Term used herein " Second-Type diabetes " also refers to non-insulin-dependent diabetes mellitus or Adult Onset's patients with type Ⅰ DM.Refer to the insulin production deficiency that Metabolic disorder causes or insulin resistant, its feature is generally fasting glucose higher than 140 milligrams/deciliter.According to this embodiment, adenine is proved acceleration glucose uptake, therefore can be used as effective therapeutic modality of the physiological situation relevant with hyperglycemia or disease.
Adenine is in the treatment of inflammatory diseases
Various AMPK activator has been proved tool anti-inflammatory function in organism.For example, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) has been proved the acute and recurrent colitis can alleviated caused by trinitro-benzene-sulfonic acid or dextran sodium sulfate in mouse model, AICAR treatment significantly reduces losing weight and slowing down inflammatory response of disease mice.In addition AICAR has obvious therapeutic effect for multiple sclerosis zootype (EAE), also reduces with the order of severity of lipopolysaccharide inducing mouse injury of lung.In the present invention, the inflammatory response that adenine suppresses lipopolysaccharide to be induced in vitro tests: under lipopolysaccharide stimulates, through the inflammatory cytohormone secretory volume of the macrophage of adenine process, comprise tumor necrosis factor α (TNF-α), be situated between white element-1 β (IL-1 β) and white element-6 (IL-6) that are situated between, and has significance to reduce compared with control group.Adenine also reduces the Transitional cell carcinomas performance amount (embodiment 4) of human macrophages because of lipopolysaccharide induction performance.In inflammatory enteropathy (IBD) mouse model of trinitro-benzene-sulfonic acid induction, give adenine treatment group other colon inflammatory cytohormone, comprise tumor necrosis factor (TNF), interferon gamma (INF γ) and be situated between white element (IL-17) all comparatively control group mice significantly reduce, and save body weight loss (embodiment 5).
Term used herein " inflammatory cytohormone " refers to the cytohormone of accelerating system inflammatory response.Term used herein " inflammatory diseases " refers to the disease relevant to inflammation, includes, but are not limited to ankylosing spondylosis, arthritis (osteoarthritis, rheumatic arthritis, psoriatic arthritis), asthma, atherosclerosis, disease, colitis, dermatitis, diverticulitis of large intestine, fibromyalgia, hepatitis, swashs hot-tempered property colon disease, systemic lupus erythematosis, nephritis, Alzheimer disease, parkinson formula disease, ulcerative colitis etc.In recent years, many reports have confirmed that AMPK is Transitional cell carcinomas upstream regulation and control person, and the protein of Transitional cell carcinomas can be suppressed to show.Conformed to previous research, inventor finds a novel AMPK activator, adenine, can effectively suppress the protein of Transitional cell carcinomas to show, it can thus be appreciated that the inflammation that adenine can suppress Transitional cell carcinomas to mediate.According to the present invention, adenine is found to suppress inflammation, therefore can be used as the treatment of physiological situation that inflammation is correlated with or disease.
Adenine is in wound healing and cicatrization
AMPK is considered to can promote Cell motility and strengthen wound healing.One AMPK activator, resveratrol, being proved can the healing of Enforcement surgery wound.Except wound healing, the formation reducing cicatrix in agglutination is the preferred object of modern medicine always.Neonate wound healing is different from the wound healing of adult, can not with the formation of cicatrix, and difference is the activation of Transitional cell carcinomas around here.In the wound healing process of adult, cyclooxygenase-2 activity can be promoted via TGF-beta, thus causes the increase that wound prostaglandin is produced.Prostaglandin has been proved the formation that can promote proliferation of fibroblast and collagen protein, and these two kinds of factors can cause cicatrization.Therefore, the activity of Transitional cell carcinomas is suppressed to be considered to effectively prevention of scar to be formed.In the present invention, adenine suppresses Fibroblast Growth (embodiment 8) and reduces the protein performance of Transitional cell carcinomas.In zootype, directly use adenine in wound and not only can strengthen wound healing and cicatrix can be reduced generating (embodiment 9).According to above-mentioned data, local application adenine can effectively be strengthened wound healing and prevent cicatrization.
Nerve degeneration
The defect of many cell mechanisms has been proved relevant with neurodegenerative disease, comprises inflammation, intracellular transport, autophagy etc.Autophagic function is to remove in cell the born of the same parents' device or protein agglomerate that lose function, and plays an important role to cell inner equilibrium.The pathogenesis of many neurodegenerative disease relates in cell or the deposition of extracellular protein agglomerate, and removes these protein agglomerates and shown the progress can improving this type of disease.In addition, autophagy approach is impaired or remove responsible autophagic protein and be proved and cause nerve degeneration.AMPK activation has confirmed to promote autophagy path.Therefore, by activating AMPK to promote autophagy path to can be used as prevention or to control the available strategy of neurodegenerative disease.AMPK activator has been proved and can have reduced kind of starch deposition via autophagy path.Give the life-span that AMPK activator-resveratrol can increase Alzheimer disease mice every day.Another AMPK activator-curcumin is also proved the potential drug that can be used as Alzheimer disease treatment.In the present invention, inventor finds that adenine is significantly strengthened autophagy activity and reduces A accumulation in neurocyte Neuro-2A, and adenine improves the cognitive function (embodiment 6,7) of Alzheimer disease disease mice in addition.According to above-mentioned discovery, the treatment that adenine can be used as neurodegenerative disease uses.
Term used herein " nerve degeneration " refers to the situation that neuronal structure or function are lost gradually.Neurodegenerative disease is neurodegenerative result, includes, but are not limited to Alzheimer disease, Parkinson's disease, Han Dingdun chorea, amyotrophic lateral sclerosis, Spinocerebellar Atrophy, myeloid muscular dystrophy etc.
Reactive chalcogen relevant disease
In biological tissue, reactive chalcogen comprises superoxide radical, hydroxyl radical free radical and hydrogen peroxide are constantly produced, and excessive reactive chalcogen is relevant with numerous disease, include, but are not limited to: nervous tissue's muscle weakness associated movement imbalance and pigmentation retinitis (NARP), MELAS syndrome, flesh is taken out the epilepsy that jumps and is merged red ragged muscle fiber disease (MERRF), Leber leber's disease (LHON), KSS syndrome, Alzheimer disease, Parkinson's disease, Han Dingdun chorea, amyotrophic lateral sclerosis, family ataxia (FA) and aging.Many research reports confirm that AMPK activator is as AICAR, under high glucose, brown Beam-at-the-eaves acid or albumin induction situation, can reduce reactive chalcogen and produce.In the present invention, adenine reduces the production (table 6) of reactive chalcogen in HUVEC cell, and the treatment that therefore adenine can be used as physiological situation that reactive chalcogen is correlated with or disease uses.
Cancer
AMPK activation suppresses Transitional cell carcinomas and mammal Rapamycin protein pathways, and this two path is the important mechanisms of growth of cancer cells.Based on Transitional cell carcinomas and mammal Rapamycin albumen to the importance of cancer, activation AMPK suppresses Transitional cell carcinomas and mammal Rapamycin protein pathways to be considered to rational strategy of cancer treatment to reach.In fact, many research reports confirm that AMPK activator interrupts cancer development, and for example, phenformin and metformin is found to suppress breast cancer tumor development and growth in the cancer mouse model of heteroplastic transplantation.In the present invention, adenine suppresses the Growth of Cells (embodiment 11) of human hepatocarcinoma cells Hep G2, mankind mastopathy cell MCF7 and colon cancer cell HT29.50% growth inhibitory concentration of adenine to Hep G2, MCF7, HT29 is respectively 544.1,537.5 and 531.9 μMs.Transplant mouse model in Hep G2, give adenine for a long time and significantly postpone tumor growth.According to the present invention, with the therapeutic modality of adenine activation AMPK, can prevent or control formation or the development of cancer.
Detailed description of the invention
Below in conjunction with specific embodiment, the invention will be further described, can better understand the present invention and can be implemented, but illustrated embodiment is not as a limitation of the invention to make those skilled in the art.
Embodiment 1
AMPK activity analysis
The analysis of adenine to AMPK phosphorylation is carried out in mouse muscle cell C2C12, Mouse fibroblasts 3T3, human hepatocarcinoma cells Hep G2, mankind mastopathy cell MCF7 and human colon cancer cells HT29 human umbilical vein endotheliocyte HUVEC, acute human Monocyte cell strain THP1, human macrophages U937, the micro-glial cell BV-2 of mice, neuroblastoma cell Neuro2A and hair papilla cell Dermal Papilla.Cell with Dulbecco's modified Eagle's medium (DMEM) containing 10% hyclone (FBS), 4mM L-glutamine, 2 mM sodium pyruvate and 1% penicillin/streptomycin (Invitrogen GibcoBRL, Carlsbad, CA, USA) in 37 ° of C, 5% CO
2cultivate under environment.3 × 10
5cell is inoculated in 6-well dish, with appointed compound process cell 30 minutes after 24 hours, and then dissolved cell analyzing with Western blot.Equal protein matter is separated with SDS-PAGE, is then transferred to polyvinylidene fluoride film.Polyvinylidene fluoride film after transfer printing is dipped to 3% bovine serum albumin after 60 minutes being dissolved in PBS buffer, add anti-phosphorylation AMPK (Thr172) antibody (1:2000 respectively, Cell signaling), anti-AMPK antibody (1:2000, Cell signaling), in 4 ° of C effects.Add corresponding two after 16 hours to resist and react 1 hour under room temperature.Tool immune response belt is detected by matter with cold light, and with egative film record signal.Analyze with TotalLab Quant software (TotalLab) after the signal scanning of gained.
The impact system that adenine activates AMPK is whole in table 1.In all test cell, adenine all significantly activates AMPK.
Table (1)
| Cell | Adenine concentration (microM) | AMPK activates (fold to control) |
| C2C12 | 1 | 1.2 |
| 10 | 1.7 | |
| 100 | 3.2 | |
| 200 | 3.9 | |
| 600 | 4.1 | |
| 3T3 | 1 | 1.1 |
| 10 | 1.5 | |
| 100 | 2.9 | |
| 200 | 4.0 | |
| 600 | 4.2 | |
| HepG2 | 1 | 1.1 |
| 10 | 2.1 | |
| 100 | 3.3 | |
| 200 | 3.8 | |
| 600 | 4.2 | |
| MCF7 | 1 | 1.2 |
| 10 | 1.6 | |
| 100 | 2.5 | |
| 200 | 3.4 | |
| 600 | 3.7 | |
| HT29 | 1 | 1.1 |
| 10 | 1.7 | |
| 100 | 2.9 | |
| 200 | 3.4 | |
| 600 | 3.8 | |
| HUVEC | 1 | 1.2 |
| 10 | 1.9 | |
| 100 | 3.2 | |
| 200 | 3.9 | |
| 600 | 4.1 | |
| THP1 | 1 | 1.2 |
| 10 | 2.2 | |
| 100 | 3.7 | |
| 200 | 4.3 | |
| 600 | 4.2 | |
| U937 | 1 | 1.1 |
| 10 | 1.3 | |
| 100 | 2.9 | |
| 200 | 3.7 | |
| 600 | 4.0 | |
| BV-2 | 1 | 1.2 |
| 10 | 1.7 | |
| 40 | 2.6 | |
| 160 | 3.2 | |
| Neuro2A | 1 | 1.2 |
| 10 | 2.1 | |
| 100 | 3.4 | |
| Dermal Papilla | 1 | 1.1 |
| 10 | 1.4 | |
| 100 | 2.1 | |
| 200 | 2.5 | |
| 600 | 2.8 |
Embodiment 2
Glucose uptake-in vitro analysis
Fluorescent glucalogue (2-NBDG, Molecular Probes) is used to analyze adenine to the impact of glucose uptake in muscle cell C2C12.C2C12 cell adds 500 μMs of fluorescent glucalogues after processing 30 minutes with the adenine of each concentration at 37 DEG C, and cultivate after 5 minutes under room temperature, cell cleans three times with Kreb-Hepes buffer solution, and fixes with 70% ethanol.The fluorescent of glucose inside cells analog is detected with fluorophotometer.
The impact system of adenine on glucose uptake is whole in table 2.Adenine significantly promotes the glucose uptake of C2C12 cell and tool concentration dependent.Data are expressed as the mean+SD of three independent experiments.
Table (2)
| Reagent | Concentration (microM) | Glucose uptake (% to control) |
| Adenine | 1 | 117±8.1 |
| 10 | 261±13.4 | |
| 100 | 315±11.9 | |
| 600 | 338±16.5 |
Embodiment 3
The anti-diabetic effect of adenine
In order to assess the impact that adenine regulates plasma glucose levels further, feeding mice using high fat diet and testing as Second-Type diabetes animal model.C57BL/6J Mouse feeder in 22 ° of C, 12 little time/circulation at night feed high fat diet (60% kcal% fat) or chow diet in not dietary restriction mode.The adenine of 0.1-50 mg/kg awards mice in 24 week age in lumbar injection mode, within after injection 1 and 3 hour, measures blood glucose value.Lumbar injection high fat diet is fed mice one day twice and is continued 6 days, and last dispensing, after 1 hour, is collected blood plasma and measures plasma glucose and triglyceride content.
Compared with feeding mice with the high fat diet of injection normal saline solution, find that adenine reduces plasma glucose amount and is greater than 30%, reduce triglyceride amount and be greater than 35%, and reduce body weight more than 15%.
Embodiment 4
The inflammatory response that adenine suppresses lipopolysaccharide to cause
In detection human macrophages, Transitional cell carcinomas albumen quality and tumor necrosis factor α (TNF-α), element-1 β (IL-1 β) and white element-6 (IL-6) secretory volume that is situated between in vain of being situated between assess adenine to the impact of inflammatory response.With 50 nM PMA process 24 hours, induction acute human Monocyte cell strain THP1 differentiation was to macrophage.THP1 macrophage stimulates 6 hours with 50 ng lipopolysaccharides containing 10 ~ 600 μMs of adenine or carrier further, then dissolved cell analyzing with Western blot.Equal protein matter is separated with SDS-PAGE, is then transferred to polyvinylidene fluoride film.Polyvinylidene fluoride film after transfer printing is dipped to 3% bovine serum albumin after 60 minutes being dissolved in PBS buffer, add anti-Transitional cell carcinomas antibody (1:1000 respectively, Cell signaling), anti-a-Actin antibody (1:5000, Cell signaling), in 4 ° of C effects.Add corresponding two after 16 hours to resist and react 1 hour under room temperature.Tool immune response belt is detected by matter with cold light, and with egative film record signal.Analyze with TotalLab Quant software (TotalLab) after the signal scanning of gained.Tumor necrosis factor α (TNF-α), element-1 β (IL-1 β) and white element-6 (IL-6) secretory volume that is situated between in vain of being situated between are analyzed with enzyme linked immunosorbent absorption method.
Adenine is whole in table 3 on immunoreactive impact system.Born of the same parents compare with control group, through the macrophage of adenine process, its cyclooxygenase-2 matter performance amount and tumor necrosis factor α (TNF-α), element-1 β (IL-1 β) and white element-6 (IL-6) secretory volume that is situated between in vain of being situated between all significantly decline.
Table (3)
| Adenine (microM) | TNFα(% to control) | IL-1β(% to control) | IL-6(% to control) | COX-2 (% to control) |
| 0 | 100±4.7 | 100±11.3 | 100±8.5 | 100±2.9 |
| 10 | 85±9.1 | 91±8.4 | 88±6.3 | 81±4.4 |
| 100 | 41±2.6 | 29±5.5 | 21±7.8 | 59±3.5 |
| 600 | 23±1.8 | 17±3.7 | 14±6.2 | 38±5.3 |
Embodiment 5
The inflammatory response that adenine suppresses trinitro-benzene-sulfonic acid to be brought out in organism
Further with inflammatory enteropathy (IBD) mouse model of trinitro-benzene-sulfonic acid induction, assessment adenine is on the impact of inflammatory response.C57BL/6J Mouse feeder in 22 ° of C, 12 little time/night circulation.The trinitro-benzene-sulfonic acid dosage progressively promoted with five: 0.5 mg, 0.75 mg, 1.0 mg, 1.25 mg and 1.5 mg are dissolved in 50% ethanol and give weekly mice 0.1mL respectively and bring out recurrent colitis.After giving trinitro-benzene-sulfonic acid for the third time, every day gives mice adenine (0.01,0.1,5 or 30 mg/kg body weight) or normal saline solution in lumbar injection mode.Within two days, sacrifice by mice after giving trinitro-benzene-sulfonic acid 5th time.The inflammatory cytohormone of colon's lysate: tumor necrosis factor (TNF), interferon gamma (INF γ) and the white element (IL-17) that is situated between are analyzed with enzyme linked immunosorbent absorption method.
Give other colon inflammatory cytohormone of adenine treatment group and comprise tumor necrosis factor (TNF), interferon gamma (INF γ) and the white element (IL-17) that is situated between, all comparatively control group mice significantly reduces, and saves body weight loss.
Embodiment 6
Kind of starch β wins peptide and autophagy activity analysis
The impact of adenine on kind of starch β victory peptide is analyzed with neuroblastoma cell Neuro2A.
Neuro2A cell with Dulbecco's modified Eagle's medium (DMEM) containing 10% hyclone (FBS), 4mM L-glutamine, 2 mM sodium pyruvate and 1% penicillin/streptomycin (Invitrogen GibcoBRL, Carlsbad, CA, USA) in 37 ° of C, 5% CO
2cultivate under environment.3 × 10
5cell is inoculated in 6-well dish, and after 24 hours, cell carries out transfection with APP695, and with adenine process cell 24 hours, then dissolved cell analyzing with Western blot.Equal protein matter is separated with SDS-PAGE, is then transferred to polyvinylidene fluoride film.Polyvinylidene fluoride film after transfer printing is dipped to 3% bovine serum albumin after 60 minutes being dissolved in PBS buffer, add anti-kind of starch β respectively and win peptide antibody (1:1000, Abcam), anti-LC3 antibody (1:1000, Cell signaling), anti-a-Actin antibody (1:5000, Cell signaling), in 4 ° of C effects.Add corresponding two after 16 hours to resist and react 1 hour under room temperature.Tool immune response belt is detected by matter with cold light, and with egative film record signal.Analyze with TotalLab Quant software (TotalLab) after the signal scanning of gained.
The impact system that adenine wins peptide and LC3-II/LC3-I ratio to kind of starch β is whole in table 4.In Neuro2A cell, adenine significance reduces kind of starch β to be won peptide amount and increases LC3-II/LC3-I ratio.Autophagic activity is represented because LC3-I is converted to LC3-II, in the cell of adenine process, the LC3-II/LC3-I ratio higher compared with control group cell, the function of reflection adenine activation autophagocytosis.
Table (4)
| Adenine (microM) | Kind of starch β wins peptide content (% of control) | LC3-II/LC3-I ratio (relative to control) |
| 0 | 100 ± 6.1 | 1.0 ± 0.1 |
| 10 | 89 ± 7.5 | 1.2 ± 0.1 |
| 20 | 63 ± 2.2 | 1.8 ± 0.3 |
| 30 | 48.1 ± 1.7 | 2.8 ± 0.2 |
| 40 | 31.7 ± 5.1 | 2.9 ± 0.2 |
| 50 | 29.4 ± 3.6 | 3.2 ± 0.3 |
Embodiment 7
Adenine wins the nerve degeneration of inducing peptide in Alzheimer disease experiment mice pattern rescue kind of starch β
Kind of starch β wins peptide 25-35 and buys from Sigma-Aldrich (St. Louis, Missouri).Victory peptide is dissolved in sterile physiological saline solution and is incubated at lower 7 days of 37 ° of C before injection.C57BL/6J Mouse feeder in 22 ° of C, 12 little time/night circulation.Become Mus with ketamine (500 mgs/kg) and xyline (100 mgs/kg) anesthesia, and be positioned over stereotaxical injection instrument.The kind of starch β of 5 nmol wins peptide 25-35 with 10 μ l injector to inject to tricorn, and side brain to be coordinate be-0.5 mm (front and back to), ± 1 mm (interior lateral) ,-2.5 mm (back of the body ventrad) are relative to anterior fontanelle.Give kind of starch β in lumbar injection mode every day and win peptide injection mice adenine or normal saline solution, adenine injected dose is 0.01,0.1,5 or 30 mg/kg of body weight, continuously injection 4 weeks.After 4 weeks, cognitive function in mice is with Mo Lisi water maze methods analyst.Water maze carries out with round pool, and platform is placed in the underwater of target quadrant to carry out hiding platform test.During 5 days hiding platform tests, each test mice is positioned over all at random as starting point in pond, 6 tests every day.Within after hiding platform tests on the 5th 1 day, carry out investigative test.When carrying out investigative test, remove hiding platform and using target quadrant opposite quadrant as starting point.With video camera record mice swimming situation of 60 seconds in labyrinth, find plateau time and swimming path with software analysis mice.
In hiding platform test, through the mice of adenine treatment, it searches out the comparatively control group mice significance minimizing of platform institute spended time.This result of the test confirms that adenine can save the impaired study of Alzheimer disease experiment mice and memory function.Moreover adenine treatment mice is in investigative test, and it stays in target and resembles line time comparatively control group mice length, proves that adenine is promoted memory and retained.
Embodiment 8
Adenine suppresses Fibroblast Growth
Mankind's fibroblast strain 3T3 with Dulbecco's modified Eagle's medium (DMEM) containing 10% hyclone (FBS), 4mM L-glutamine, 2 mM sodium pyruvate and 1% penicillin/streptomycin (Invitrogen GibcoBRL, Carlsbad, CA, USA) in 37 ° of C, 5% CO
2cultivate under environment.In Cell Growth Assays, 1 × 10
5cell is inoculated in 6-well dish, after 24 hours, with the adenine process cell 72 hours of prescribed concentration, calculates the cell number of survival.Cell is separated with trypsin-EDTA and dyes with trypan blue, with blood cell calculator calculating survivaling cell number.
The impact system of adenine on 3T3 Growth of Cells is whole in table 5.As seen from the results in Table 5, adenine significantly suppresses 3T3 Growth of Cells and tool dose dependent.Data are expressed as the mean+SD of three independent experiments.
Table (5)
| Adenine (microM) | Cell number (% to control) |
| 0 | 100±4.3 |
| 10 | 91±2.7 |
| 50 | 73±8.1 |
| 100 | 64±5.3 |
| 200 | 48±2.8 |
| 500 | 33±6.4 |
| 1000 | 27±11.3 |
Embodiment 9
Adenine is strengthened wound healing and is reduced cicatrization
C57BL/6J Mouse feeder in 22 ° of C, 12 little time/night circulation.Become 12 week age Mus with ketamine (500 mgs/kg) and xyline (100 mgs/kg) anesthesia, manufacture wound in mouse back with 6-mm skin sampling device.After forming wound, use 10 ~ 1200 μMs of adenine or saline solution in wound.Skin wound is then fixed with semi permeability slide wound dressing.Mice is sacrificed after 14 days with adenine or physiological saline water treatment.Cicatrization is with Masson ' s trichrome staining analysis (tissue is fixed with 4% paraformaldehyde).
After process in 14 days, comparatively control group is fast for the healing rate of adenine process wound, and according to tissue staining analysis, with the wound regenerating tissues of adenine process, its cicatrix is significantly little compared with control group wound.
Embodiment 10
Adenine reduces reactive chalcogen and generates
Human umbilical vein's endotheliocyte HUVEC with Dulbecco's modified Eagle's medium (DMEM) containing 10% hyclone (FBS), 4mM L-glutamine, 2 mM sodium pyruvate and 1% penicillin/streptomycin (Invitrogen GibcoBRL, Carlsbad, CA, USA) in 37 ° of C, 5% CO
2cultivate under environment.2 × 10
4cell is inoculated in the black dish of 96-well, after 24 hours, culture medium is replaced as the DMEM culture medium containing 5.6 or 30 mM glucoses and adds the adenine of prescribed concentration.Process after 24 hours, in cell, reactive chalcogen is detected with H2DCF-DA.Cell, with after PBS buffer solution for cleaning 1 time, is incubated at 100 μMs of DCF, 37 ° of C 30 minutes.DCF fluorescent analyzes (excitation wavelength: 485 nm with disc type florescence analysis instrument; Scattering wavelength: 530 nm).
The impact system that adenine generates reactive chalcogen is whole in table 6.The reactive chalcogen that adenine significance reduces high glucose induction generates and tool dose dependent.
Table (6)
| Adenine (microM) | Glucose (mM) | Reactive chalcogen generates (% of 5.6 mM glucose) |
| 0 | 30 | 275± 8.1 |
| 10 | 30 | 211± 4.3 |
| 100 | 30 | 116± 1.7 |
| 200 | 30 | 38.1± 2.9 |
| 600 | 30 | 21.7± 3.1 |
| 1200 | 30 | 22.4± 2.5 |
Embodiment 11
Growth of cancer cells inhibition test
The impact of adenine on growth of cancer cells is carried out with human hepatocarcinoma cells Hep G2, mankind mastopathy cell MCF7 and human colon cancer cells HT29.Cell with Dulbecco's modified Eagle's medium (DMEM) containing 10% hyclone (FBS), 4mM L-glutamine, 2 mM sodium pyruvate and 1% penicillin/streptomycin (Invitrogen GibcoBRL, Carlsbad, CA, USA) in 37 ° of C, 5% CO
2cultivate under environment.1 × 10
5cell is inoculated in 6-well dish, after 24 hours, with the adenine process cell 72 hours of prescribed concentration, calculates the cell number of survival.Cell is separated with trypsin-EDTA and dyes with trypan blue, with blood cell calculator calculating survivaling cell number.
50% growth inhibitory concentration of adenine to Hep G2, MCF7, HT29 is respectively 544.1,537.5 and 531.9 μMs.
Embodiment 12
Tumor growth assay
Human hepatocarcinoma cells Hep G2 with Dulbecco's modified Eagle's medium (DMEM) containing 10% hyclone (FBS), 4mM L-glutamine, 2 mM sodium pyruvate and 1% penicillin/streptomycin (Invitrogen GibcoBRL, Carlsbad, CA, USA) in 37 ° of C, 5% CO
2cultivate under environment.5 × 10
6cell is injected to NOD-SCID mice in 8 week age with injected s. c.After transplanting, every day gives the adenine of mice 5,20,50 mgs/kg of body weight in lumbar injection mode, within every 3 days, measures tumor size.Transplant latter 14 days, compared to control group mice, give the growth that adenine significantly delays tumor.
The above embodiment is only that protection scope of the present invention is not limited thereto in order to absolutely prove the preferred embodiment that the present invention lifts.The equivalent alternative or conversion that those skilled in the art do on basis of the present invention, all within protection scope of the present invention.Protection scope of the present invention is as the criterion with claims.
Claims (11)
1. for activating a compound of AMPK, it is characterized in that, it is adenine and/or its pharmaceutically acceptable salt.
2. compound described in claim 1 treats the application of the medicine of disease or the physiological situation that can be improved by AMPK activator as preparation.
3. compound described in claim 1 treats the application of the medicine of inflammatory physiological situation or disease as preparation.
4. compound described in claim 1 is as the application of preparation prevention or treatment pre-diabetes, Second-Type diabetes, metabolic syndrome a kind of or physiological situation of its combination or the medicine of disease.
5. compound described in claim 1 is as the application of preparation prevention or treatment pre-diabetes, Second-Type diabetes, metabolic syndrome a kind of or physiological situation of its combination or the medicine of disease.
6. compound described in claim 1 is as the application preparing the medicine preventing or treat Alzheimer disease.
7. compound described in claim 1 treats the application of the medicine of disease or the physiological situation that can be improved by autophagocytosis as preparation.
8. compound described in claim 1 is as the application of medicine preparing wound healing process and suppress cicatrization.
9. compound described in claim 1 strengthens the application of the medicine of wound healing as preparation.
10. compound described in claim 1 is as the application of the medicine of the cell of protecting and treating receptor 1 activity chalcogen to injure in preparation mammal.
Compound described in 11. claim 1 is as the application preparing prevention or Therapeutic cancer medicine.
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