CN104510736B

CN104510736B - AMPK-activating compounds and uses thereof

Info

Publication number: CN104510736B
Application number: CN201310444518.XA
Authority: CN
Inventors: 邱壬乙; 陈翰民; 郭正宜; 林俊材; 黄纯芳
Original assignee: Huaan Medical Co ltd
Current assignee: Huaan Medical Co ltd
Priority date: 2013-09-26
Filing date: 2013-09-26
Publication date: 2020-09-29
Anticipated expiration: 2033-09-26
Also published as: CN111939162A; CN104510736A; CN111939161A; CN111920817A

Abstract

The invention discloses a compound for activating AMPK, which is adenine and/or a pharmaceutically acceptable salt thereof. Also disclosed are uses of the compounds for activating AMPK (AMP-activated protein kinase), and for preventing or treating a physiological condition or disease, and thus a physiological condition or disease ameliorated by AMPK, in a mammal.

Description

AMPK-activating compounds and uses thereof

Technical Field

The present invention relates to adenine, which is useful for activating AMPK (AMP-activated protein kinase) and preventing or treating physiological conditions or diseases using the same.

Background

AMPK is well defined as a sensor of cellular energy and a response to energy demand. AMPK isThe isotrimer consists of a catalytic α subunit, a regulatory β, a gamma subunit, all of which are highly conserved in eukaryotes AMPK activation is by its upstream kinases such as LKB1, a calcium ion/calcitonin-carrying dependent protein phosphokinase (Ca)²⁺Calmodulindendependent kinase) and TAK1 phosphorylate the α subunit with a conserved 172 th threonine residue, which leads to high AMP/ATP ratios due to physiological or pathological stress and also activates ampk, which, upon activation, promotes catabolic pathways and inhibits anabolism, restoring cellular energy balance by reducing ATP consumption and promoting ATP production.

AMPK, a regulator of energy metabolism balance, is considered as a potential drug marker for metabolic syndrome, including type ii diabetes, cardiovascular disease, fatty liver, and the like. Many metabolic syndromes are associated with insulin resistance. Insulin resistance is a pathological condition in which cells fail to respond to insulin and excess glucose in the blood cannot be removed to skeletal muscle or adipose tissue. In muscle cells, AMPK activation increases the expression of glucose transporter (GLUT4) in a non-insulin dependent manner through transcriptional regulation and induces the translocation of GLUT4 to the cell membrane resulting in increased rates of glucose uptake by the cells. AMPK activation also inhibits fatty acid and cholesterol synthesis by inhibiting acetyl-CoA carboxylase (acetyl-CoA carboxylase) and hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase), respectively. In addition, AMPK activation results in the inhibition of several transcription factors, including SREBP-1c, ChREBP and HNF-4a, and decreases protein expression of enzymes involved in the regulation of fatty acid synthesis and carbohydrate neogenesis. The above mentioned studies have all supported that AMPK is the target for the treatment of metabolic syndrome, in particular diabetes.

In addition to the regulation of energy metabolism balance, AMPK is also involved in the regulation of several cellular mechanisms, including inflammatory response, cell growth, apoptosis, autophagy, aging, and differentiation. Many studies have shown that AMPK is an inhibitor of inflammatory responses. AMPK activation suppresses inflammatory responses by inhibiting the signaling of nuclear transcription factor (NF- κ B). The signal transmission of the nuclear transcription factor is the main path for activating innate immunity and acquired immunity, and after AMPK is activated, the transcription activity of the nuclear transcription factor is inhibited by stimulating SIRT1, Forkhead box O (FoxO) or peroxisome promoter-activated receptor co-activator 1 alpha (PGC1 alpha) so as to achieve the effect of inhibiting the inflammatory response. In addition, several laboratories have also demonstrated that AMPK activation inhibits the protein expression of cyclooxygenase-2 (COX-2). Cyclooxygenase-2 is an inducible enzyme that is regulated by inflammatory cytokines and growth factors and functions to convert arachidonic acid into prostaglandins resulting in inflammatory reactions and pain, and thus inhibition of cyclooxygenase activity or expression has been shown to have anti-inflammatory effects.

Several AMPK activators have been demonstrated to have anti-inflammatory activity in vivo. For example, 5-aminoimidazole-4-carboxamide riboside (AICAR) has been shown in the mouse model to alleviate acute and recurrent colitis caused by trinitrobenzene sulfonic acid or sodium dextran sulfate, AICAR treatment significantly reduced the weight loss and slowed the inflammatory response in diseased mice. In addition, AICAR has a significant therapeutic effect on the human multiple sclerosis animal model (EAE), and also reduces the severity of lung injury in mice induced by lipopolysaccharide.

Dysregulation of cellular signaling pathways may lead to abnormal growth of cells, ultimately leading to cancer. Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates cell growth and autophagy. Deregulated activity of mammalian rapamycin-targeted protein signaling pathways has been found in many different cancers, and therefore mammalian inhibitors of rapamycin-targeted proteins are considered potential agents for cancer therapy. Numerous studies have demonstrated that AMPK phosphorylates the protein of the turbous sylvestris complex 2 (TSC2) and Raptor achieves inhibition of the mammalian target protein pathway of rapamycin. Various AMPK activators including AICAR, metformin, phenformin have been shown to inhibit mammalian rapamycin-targeted protein signaling pathways, and inhibit cancer cell growth. In addition, AMPK activation induces autophagy through inhibition of mammalian rapamycin-targeted protein complex-1. Since AMPK inhibits the decrease in phosphorylation of 757 serine on the mammalian rapamycin target protein complex-1, Ulk1, phosphorylation of 317 and 777 serine by AMPK is followed by the initiation of autophagy Ulk1 by AMPK phosphorylation.

In summary, AMPK is considered to be a good therapeutic target for a number of human diseases or pathological conditions, including inflammatory diseases, wound healing, neurodegeneration, cancer, oxidative stress and cardiovascular diseases. In fact, AMPK activators have been used in clinical trials for at least 24 types of diseases, including bacterial and fungal diseases, behavioral and psychological disorders, hematological and lymphatic diseases, cancer, tumors, digestive diseases, otorhinolaryngological diseases, ophthalmic diseases, glandular and hormone-related diseases, cardiovascular diseases, immune system diseases, mouth and dental diseases, muscle, bone, cartilage diseases, nervous system diseases, nutritional and metabolic diseases, respiratory diseases, skin and connective tissue diseases, wound healing, and the like.

Disclosure of Invention

The present invention provides a novel AMPK activator-adenine and the use of the compound for the prevention or treatment of diseases.

The present invention provides a compound for activating AMPK, which is adenine and/or a pharmaceutically acceptable salt thereof.

The present invention provides the use of the above compounds for the preparation of a medicament for the treatment of diseases or physiological conditions which can be ameliorated by AMPK activators.

The invention provides the use of the above compounds for the preparation of a medicament for the treatment of an inflammatory physiological condition or disease.

The invention provides application of the compound in preparing a medicament for preventing or treating physiological conditions or diseases of one or a combination of pre-diabetes, second diabetes and metabolic syndrome.

The invention provides application of the compound in preparing a medicament for preventing or treating Alzheimer's disease.

The invention provides the use of the above compounds for the preparation of a medicament for the treatment of a disease or physiological condition ameliorated by autophagy.

The invention provides application of the compound in preparing a medicament for inhibiting scar formation in a wound healing process.

The invention provides the use of the above compounds for the preparation of a medicament for enhancing wound healing.

The invention provides the use of the above compounds for the preparation of a medicament for the protection and treatment of cells damaged by reactive oxygen species in a mammal.

The invention provides application of the compound in preparing a medicament for preventing or treating cancer.

According to an embodiment of the present invention, there is provided a novel AMPK activator, adenine, which activates AMPK intracellularly, thereby preventing or treating physiological conditions or diseases that can be improved by AMPK in mammals.

According to an embodiment of the present invention, there is provided a method for reducing blood glucose by activating AMPK, thereby preventing or treating diseases including metabolic syndrome, pre-diabetes, type II diabetes, and insulin resistance, wherein an effective amount of adenine and/or a pharmaceutically acceptable salt thereof is administered to a mammal in need of such treatment.

According to an embodiment of the present invention, there is provided a method for preventing or treating an inflammatory condition or disease by activating AMPK to prevent inflammation, wherein an effective amount of adenine and/or a pharmaceutically acceptable salt is administered to a mammal in need of such treatment.

According to an embodiment of the present invention, there is provided a method for inhibiting fibroblast growth by activating AMPK, thereby preventing scar tissue formation during wound healing.

According to an embodiment of the present invention, there is provided a method of enhancing wound healing, wherein an effective amount of adenine and/or a pharmaceutically acceptable salt is administered to a mammal in need of such treatment.

According to an embodiment of the present invention, there is provided a method of inhibiting the production of Reactive Oxygen Species (ROS) to protect or treat cells of a mammal from reactive oxygen species damage, wherein an effective amount of adenine and/or a pharmaceutically acceptable salt is administered to a mammal in need of such treatment.

According to an embodiment of the present invention, there is provided a method of inhibiting the growth of cancer cells, thereby preventing or treating cancer, wherein an effective amount of adenine and/or a pharmaceutically acceptable salt is administered to a mammal in need of such treatment.

The present invention relates to adenine which is suitable for activating AMPK and using adenine for preventing or treating physiological conditions or diseases, including: pre-diabetes, insulin resistance, type II diabetes, metabolic syndrome, obesity, inflammation, wound healing, Alzheimer's disease, cancer, oxidative stress and cardiovascular disease.

The invention discovers that adenine is a novel AMPK activator and has various biological functions. In recent years, AMPK activation has been shown to be useful in the prevention and treatment of diseases such as pre-diabetes, insulin resistance, type ii diabetes, metabolic syndrome, obesity, inflammation, wound healing, alzheimer's disease, cancer, oxidative stress, cardiovascular disease and promotion of wound healing. It is believed that this effect may be attributed to, but is not limited to, decreased cyclooxygenase-2 expression, inhibition of Reactive Oxygen Species (ROS) production, and increased glucose uptake activity, resulting from AMPK activation.

Prospective indications

Based on the results of the present study (see examples), adenine can be used as a therapeutic agent for various physiological conditions or diseases by activating AMPK. Exemplary guidance and evidence for the desired indication is provided below.

Adenine for treating hyperglycemia, prediabetes, insulin resistance and second diabetes

It has recently been reported that AMPK activators including metformin, a769662, AICAR lower plasma glucose concentrations in diabetic or obese mouse models. In the present invention, adenine at 1. mu.M-600. mu.M significantly increased glucose uptake by muscle cells C2C12 (Table 2). Mice fed with high-fat diet were further evaluated as a second type diabetic animal model for the effect of adenine on regulation of plasma glucose concentration. Adenine administration significantly reduced plasma glucose by more than 30% and reduced plasma triglycerides by more than 35% and a weight loss of more than 15% in mice fed with the high fat diet as compared to mice fed with the control high fat diet (example 3). The term "hyperglycemic" as used herein refers to a physiological condition characterized by blood glucose levels greater than 126 mg/dl. As used herein, the term "pre-diabetes" refers to a physiological condition characterized by fasting glucose of greater than 100 mg/dl but less than 140 mg/dl. The term "insulin resistance" as used herein refers to a physiological condition in which the whole body or tissues including liver, skeletal muscle, adipose tissue fail to respond to insulin. The term "type II diabetes" as used herein also refers to non-insulin dependent diabetes mellitus or adult onset diabetes. Refers to insulin deficiency or insulin resistance caused by metabolic disorders, which is generally characterized by fasting glucose levels above 140 mg/dl. According to this example, adenine is demonstrated to accelerate glucose uptake and therefore may be an effective treatment for physiological conditions or diseases associated with hyperglycemia.

Treatment of inflammatory diseases with adenine

Various AMPK activators have been demonstrated to have anti-inflammatory functions in the organism. For example, 5-aminoimidazole-4-carboxamide riboside (AICAR) has been shown in the mouse model to alleviate acute and recurrent colitis caused by trinitrobenzene sulfonic acid or sodium dextran sulfate, AICAR treatment significantly reduced the weight loss and slowed the inflammatory response in diseased mice. In addition, AICAR has a significant therapeutic effect on the human multiple sclerosis animal model (EAE), and also reduces the severity of lung injury in mice induced by lipopolysaccharide. In the present invention, adenine inhibits lipopolysaccharide-induced inflammatory responses in an in vitro assay: the secretion of inflammatory cytokines including tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) by adenine-treated macrophages was significantly reduced compared to the control group under lipopolysaccharide stimulation. Adenine also reduces cyclooxygenase-2 expression in human macrophages due to lipopolysaccharide-induced expression (example 4). In the trinitrobenzenesulfonic acid-induced Inflammatory Bowel Disease (IBD) mouse model, colonic inflammatory cytokines including Tumor Necrosis Factor (TNF), interferon gamma (INF γ), and interleukin (IL-17) were administered to the adenine-treated group significantly less than control mice, and saved body weight loss (example 5).

The term "inflammatory cytokines" as used herein refers to cytokines that promote the systemic inflammatory response. The term "inflammatory disease" as used herein refers to diseases associated with inflammation, including, but not limited to, ankylosing spondylitis, arthritis (osteoarthritis, rheumatoid arthritis, psoriatic arthritis), asthma, atherosclerosis, crohn's disease, colitis, dermatitis, diverticulitis of the large intestine, fibromyalgia, hepatitis, irritable colon disease, systemic lupus erythematosus, nephritis, alzheimer's disease, parkinson's disease, ulcerative colitis, and the like. In recent years, a number of reports have demonstrated that AMPK is an upstream regulator of cyclooxygenase-2 and inhibits the protein expression of cyclooxygenase-2. In line with previous studies, the inventors found that a novel AMPK activator, adenine, is effective in inhibiting cyclooxygenase-2 protein expression, and thus it was known that adenine inhibits cyclooxygenase-2 mediated inflammation. According to the present invention, adenine has been found to inhibit inflammation and thus may be useful as a treatment for physiological conditions or diseases associated with inflammation.

Adenine for wound healing and scar formation

AMPK is thought to promote cell motility and enhance wound healing. An AMPK activator, resveratrol, has been shown to enhance healing of surgical wounds. In addition to wound healing, reducing scar formation during the healing process has been a primary goal of modern medicine. Neonatal wound healing differs from adult wound healing in that it is not accompanied by scar formation, a difference being the activation of cyclooxygenase-2. In the wound healing process in adults, cyclooxygenase-2 activity is elevated via TGF-beta, resulting in increased prostaglandin production at the wound. Prostaglandins have been shown to promote fibroblast proliferation and collagen formation, both of which can lead to scar formation. Therefore, inhibition of cyclooxygenase-2 activity is considered to be effective in preventing scar formation. In the present invention, adenine inhibits fibroblast growth (example 8) and reduces the protein expression of cyclooxygenase-2. In animal models, adenine administration directly to the wound not only enhances wound healing but also reduces scar formation (example 9). According to the above data, topical application of adenine is effective in enhancing wound healing and preventing scar formation.

Neurodegeneration

Defects in many cellular mechanisms have been shown to be associated with neurodegenerative diseases, including inflammation, intracellular trafficking, autophagy, and the like. Autophagy functions to remove dysfunctional organelles or protein masses within cells and plays an important role in intracellular balance. The pathogenesis of many neurodegenerative diseases involves the deposition of intracellular or extracellular protein masses, and removal of these protein masses has been shown to improve the progression of such diseases. In addition, impairment of the autophagy pathway or removal of proteins responsible for autophagy has been shown to lead to neurodegeneration. AMPK activation has been shown to promote the autophagy pathway. Thus, promoting the autophagy pathway by activating AMPK may be an effective strategy for preventing or controlling neurodegenerative diseases. AMPK activators have been shown to reduce amyloidogenic deposition via the autophagic pathway. Daily administration of AMPK activator-resveratrol increases the longevity of alzheimer's mice. Another AMPK activator, curcumin, has also been shown to be a potential drug for the treatment of alzheimer's disease. In the present invention, the inventors found that adenine significantly potentiates autophagy activity and reduces a accumulation in neural cell Neuro-2A, and furthermore adenine improves cognitive function in mice with alzheimer's disease (examples 6, 7). Based on the above findings, adenine is useful as a therapeutic agent for neurodegenerative diseases.

The term "neurodegeneration" as used herein refers to the gradual loss of neuronal structure or function. Neurodegenerative diseases are the result of neurodegeneration and include, but are not limited to, Alzheimer's disease, Parkinson's disease, Handington's disease, amyotrophic lateral sclerosis, spinocerebellar atrophy, spinal muscular atrophy, and the like.

Reactive oxygen species related diseases

Reactive oxygen species including superoxide radical, hydroxyl radical and hydrogen peroxide are continuously produced in biological tissues and excess reactive oxygen species are associated with a number of diseases including, but not limited to, nervous tissue muscle weakness with movement disorders and retinitis pigmentosa (NARP), MELAS syndrome, muscular withdrawal epilepsy with redness \35124, regiment muscle fibrosis (MERRF), Leber hereditary optic atrophy (LHON), KSS syndrome, alzheimer's disease, parkinson's disease, handington's disease, amyotrophic lateral sclerosis, Friedreich's Ataxia (FA) and aging. Many research reports demonstrate that AMPK activators such as AICAR, reduce reactive oxygen species production in high glucose, palmitic acid or albumin induced conditions. In the present invention, adenine reduces the production of reactive oxygen species in HUVEC cells (table 6), and therefore adenine may be used as a treatment for reactive oxygen species-related physiological conditions or diseases.

Cancer treatment

AMPK activation inhibits cyclooxygenase-2 and the mammalian rapamycin target protein pathway, both of which are important mechanisms of cancer cell growth. Based on the importance of cyclooxygenase-2 and mammalian target of rapamycin proteins for cancer, activation of AMPK to inhibit the cyclooxygenase-2 and mammalian target of rapamycin protein pathways is considered a rational cancer treatment strategy. Indeed, many studies report that AMPK activators interrupt cancer development, for example, phenoformin and metformin were found to inhibit breast cancer tumor development and growth in xenografted cancer mouse models. In the present invention, adenine inhibits the cell growth of human hepatoma cell Hep G2, human breast cancer cell MCF7 and colon cancer cell HT29 (example 11). The 50% growth inhibitory concentrations of adenine to Hep G2, MCF7, HT29 were 544.1, 537.5 and 531.9. mu.M, respectively. In Hep G2 transplanted mouse model, long-term adenine administration significantly delayed tumor growth. According to the present invention, the formation or development of cancer can be prevented or controlled in a therapeutic manner in which AMPK is activated by adenine.

Detailed Description

The present invention is further described with reference to specific examples to enable those skilled in the art to better understand the present invention and to practice the same, but the examples are not intended to limit the present invention.

Example 1

AMPK Activity assay

The analysis of the effect of adenine on AMPK phosphorylation was performed on mouse muscle cell C2C12, mouse fibroblast 3T3, human hepatoma cell Hep G2, human breast cancer cell MCF7 and human colon cancer cell HT29 human umbilical vein endothelial cell HUVEC, human acute monocyte cell line THP1, human macrophage U937, mouse microneuropal cell BV-2, neuroblastoma cell Neuro2A and hair Papilla cell Dermal Papila. The cells were purified from Dulbecco 'modified Eagle's medium (DMEM) containing 10% Fetal Bovine Serum (FBS), 4mM L-glutamine, 2 mM Sdochium pyroltate and 1% penicillin/streptomycin (Invitrogen GibcoBRL, Carlsbad, Calif., USA) at 37 ℃ in 5% CO₂Culture under ambient conditions 3 × 10⁵Cells were seeded in 6-well plates and 24 hours later cells were treated with the indicated compounds for 30 minutes, followed by lysis and analysis by western blotting. Equivalent proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane. After the transferred polyvinylidene fluoride membrane is soaked in 3% bovine serum albumin dissolved in PBS buffer solution for 60 minutes, anti-phosphorylation AMPK (Thr172) antibody (1:2000, Cell signaling) and anti-AMPK antibody (1:2000, Cell signaling) are respectively added to act at 4 ℃. After 16 hours, the corresponding secondary antibody was added and reacted at room temperature for 1 hour. The immunoreactive band is detected by a luminescent substrate and the signal is recorded by a negative. The resulting signals were scanned and analyzed with totalalab Quant software (totalalab).

The effect of adenine on AMPK activation is summarized in table 1. In all cells tested, adenine significantly activates AMPK.

Watch (1)

Cells	Adenine concentration (microM)	AMPK activation (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

Example 2

Glucose uptake-in vitro assay

The effect of adenine on glucose uptake was analyzed in muscle cells C2C12 using fluorescent glucose analogs (2-NBDG, Molecular Probes). After C2C12 cells were treated with adenine at each concentration at 37 ℃ for 30 minutes, 500. mu.M of a fluorescent glucose analog was added, and after culturing at room temperature for 5 minutes, the cells were washed three times with Kreb-Hepes buffer solution and fixed with 70% ethanol. The fluorescence of intracellular glucose analogs was detected by fluorescence photometry.

The effect of adenine on glucose uptake is summarized in table 2. Adenine significantly promotes glucose uptake by C2C12 cells and is concentration dependent. Data are presented as mean ± standard deviation of three independent experiments.

Watch (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

Example 3

Antidiabetic effects of adenine

To further assess the effect of adenine on the regulation of plasma glucose levels, mice fed high fat diet were tested as an animal model of type ii diabetes. C57BL/6J mice were housed at 22 ℃, on a 12 hour day/night cycle and fed either high fat diet (60% kcal% fat) or normal diet without restriction. 0.1-50 mg/kg adenine was administered intraperitoneally to 24-week-old mice, and blood glucose values were measured 1 and 3 hours after injection. Mice were fed with high fat diet by intraperitoneal injection twice a day for 6 days, and 1 hour after the last administration, plasma was collected and plasma glucose and triglyceride contents were measured.

Compared with mice fed with high-fat feed supplemented with physiological saline, adenine was found to reduce plasma glucose by more than 30%, triglyceride by more than 35%, and body weight by more than 15%.

Example 4

Adenine inhibits inflammatory responses caused by lipopolysaccharide

The effect of adenine on the inflammatory response was assessed by measuring the amount of cyclooxygenase-2 protein and the amount of tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) secreted in human macrophages. Human acute monocyte cell line THP1 was induced to differentiate into macrophages by treatment with 50 nM PMA for 24 hours. THP1 macrophages were further stimulated with 50 ng lipopolysaccharide containing 10-600 μ M adenine or vehicle for 6 hours, followed by cell lysis and western blot analysis. Equivalent proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane. After the transferred polyvinylidene fluoride membrane is soaked in 3% bovine serum albumin dissolved in PBS buffer solution for 60 minutes, anti-cyclooxygenase-2 antibody (1:1000, Cell signaling) and anti-motor protein antibody (1:5000, Cell signaling) are respectively added for acting at 4 ℃. After 16 hours, the corresponding secondary antibody was added and reacted at room temperature for 1 hour. The immunoreactive band is detected by a luminescent substrate and the signal is recorded by a negative. The resulting signals were scanned and analyzed with totalalab Quant software (totalalab). The secretion of TNF alpha (TNF-alpha), IL-1 beta (IL-1 beta) and IL-6 (IL-6) was analyzed by enzyme linked immunosorbent assay.

The effect of adenine on the immune response is summarized in Table 3. Compared with the control histocyte, the expression level of the cyclooxygenase-2 protein and the secretion levels of tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) of the macrophage treated by adenine are all obviously reduced.

Watch (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

Example 5

Inhibition of trinitrobenzenesulfonic acid-induced inflammatory responses in organisms by adenine

The effect of adenine on the inflammatory response was further evaluated in the trinitrobenzenesulfonic acid-induced Inflammatory Bowel Disease (IBD) mouse model. C57BL/6J mice were housed at 22 ℃ for a 12 hour day/night cycle. At five escalated trinitrobenzenesulfonic acid doses: 0.5 mg, 0.75 mg, 1.0 mg, 1.25 mg and 1.5 mg, dissolved in 50% ethanol, were administered to mice 0.1mL per week, respectively, to induce recurrent colitis. Mice were given adenine (0.01,0.1, 5 or 30 mg/kg body weight) or saline daily by intraperitoneal injection after the third administration of trinitrobenzenesulfonic acid. Mice were sacrificed two days after the fifth administration of trinitrobenzene sulfonic acid. Inflammatory cytokines of colonic tissue lysate: tumor Necrosis Factor (TNF), interferon gamma (INF gamma) and interleukin (IL-17) were analyzed by enzyme-linked immunosorbent assay.

Colonic inflammatory cytokines including Tumor Necrosis Factor (TNF), interferon gamma (INF gamma) and interleukin (IL-17) were administered to the adenine treatment group, all significantly reduced compared to the control mice, and saved the weight loss.

Example 6

Amyloid beta peptide and autophagy activity assay

Neuroblastoma cell Neuro2A was used to analyze the effect of adenine on amyloid β peptide.

Neuro2A cells were treated with Dulbecco's Modified Eagle's Medium (DMEM) containing 10% Fetal Bovine Serum (FBS), 4mM L-glutamine, 2 mM sodium sulfate and 1% penicilin/streptomycin (Invitrogen GibcoBRL, Carlsbad, Calif., USA) at 37 ℃ and 5% CO₂Culture under ambient conditions 3 × 10⁵Cells were seeded on 6-well plates and 24 hours later, cells were transfected with APP695 and treated with adenine for 24 hours, then lysed and analyzed by Western blot method equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membrane after transfer polyvinylidene fluoride membrane was soaked in 3% bovine serum albumin in PBS buffer for 60 minutes, anti-amyloidogenic β peptide antibody (1:1000, Abcam), anti-LC 3 antibody (1:1000, Cell signaling), anti-automotive protein antibody (1:5000, Cell signaling) were added, respectively, at 4 ℃ after 16 hours, the corresponding secondary antibody was added and reacted at room temperature for 1 hour, immunoreactive bands were detected with luminescent substances and signals were recorded with negative films.

The effect of adenine on the ratio of amyloid β peptide to LC3-II/LC3-I is summarized in Table 4. In Neuro2A cells, adenine significantly reduced the amount of amyloid β peptide and increased the LC3-II/LC3-I ratio. Since the transition from LC3-I to LC3-II represents autophagic activity, the higher LC3-II/LC3-I ratio in adenine-treated cells compared to control cells reflects the function of adenine to activate autophagy.

Watch (4)

Adenine (microM)	Amyloid β 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

Example 7

Mode of adenine in Alzheimer's disease experimental mouse for rescuing amyloidogenic beta peptide-induced neurodegeneration

Amyloidogenic beta peptides 25-35 were purchased from Sigma-Aldrich (St. Louis, Missouri). Peptides were dissolved in sterile physiological saline and incubated at 37 ℃ for 7 days prior to injection. C57BL/6J mice were housed at 22 ℃ for a 12 hour day/night cycle. The adult mice were anesthetized with ketamine (500 mg/kg) and xyline (100 mg/kg) and placed in a stereotaxic injection apparatus. 5 nmol of starch-like beta peptide 25-35 was injected into the lateral ventricle of the brain with a 10. mu.l syringe, the lateral brain being coordinated by-0.5 mm (antero-posterior), + -1 mm (medio-lateral), -2.5 mm (dorsoventral) relative to the anterior chimney. The amyloidogenic beta peptide is injected into mice with adenine or normal saline by intraperitoneal injection every day, the injection dosage of the adenine is 0.01,0.1, 5 or 30 mg/kg of body weight, and the injection is continuously carried out for 4 weeks. After 4 weeks, the mouse cognitive function was analyzed by the morris water maze method. The water maze is carried out with circular pond, and the platform is arranged in the surface of water of target quadrant in order to hide the platform test. During the 5-day hidden platform test period, mice were randomly placed in the pool as the starting point for each test, and 6 tests were performed daily. Exploratory tests were performed 1 day after the 5 day hidden platform test. When the exploratory test is carried out, the hidden platform is removed, and the opposite quadrant of the target quadrant is used as a starting point. The video camera is used for recording the condition that the mouse swims for 60 seconds in the maze, and software is used for analyzing the time for searching the platform and the swimming path of the mouse.

In the hidden platform test, the time spent in finding the platform was significantly less for the adenine-treated mice than for the control mice. The test result proves that adenine can save the impaired learning and memory functions of the mice in the Alzheimer's disease experiment. Furthermore, adenine-treated mice remained longer on the target image line than control mice in the exploratory test, demonstrating that adenine enhances memory retention.

Example 8

Inhibition of fibroblast growth by adenine

Human fibroblast 3T3 was prepared by Dulbecco's Modified Eagle's Medium (DMEM) containing 10% Fetal Bovine Serum (FBS), 4mM L-glutamine, 2 mM sodium sulfateAnd 1% penicillin/streptomycin (Invitrogen GibcoBRL, Carlsbad, Calif., USA) at 37 ℃ in 5% CO₂Culture in the Environment in cell growth test, 1 × 10⁵Cells were seeded in 6-well plates and 24 hours later, cells were treated with adenine at the indicated concentration for 72 hours and the number of surviving cells was counted. Cells were detached with trypsin-EDTA and stained with trypan blue, and viable cell number was counted with a hemocytometer.

The effect of adenine on 3T3 cell growth is summarized in table 5. As can be seen from the results in table 5, adenine significantly inhibited the growth of 3T3 cells and was dose-dependent. Data are presented as mean ± standard deviation of three independent experiments.

Watch (5)

Adenine (microM)	Number of cells (% 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

Example 9

Adenine enhances wound healing and reduces scar formation

C57BL/6J mice were housed at 22 ℃ for a 12 hour day/night cycle. 12-week-old adult mice were anesthetized with ketamine (500 mg/kg) and xyline (100 mg/kg), and wounds were made on the backs of the mice with a 6-mm skin sampler. After the wound is formed, 10 to 1200. mu.M adenine or saline is applied to the wound. The skin wound was then secured with a semi-permeable transparent sheet wound dressing. Mice were sacrificed 14 days after treatment with adenine or saline. Scar formation was analyzed by Masson's trichrome staining (tissue fixed at 4% parafumaldehyde).

After 14 days of treatment, the healing rate of the adenine-treated wounds was faster than that of the control group, and the wounds treated with adenine regenerated tissue with significantly less scars than the control group wounds according to tissue staining analysis.

Example 10

Adenine reduces reactive oxygen species formation

HUVEC human umbilical vein endothelial cells HUVEC were treated with Dulbecco's Modified Eagle's Medium (DMEM) containing 10% Fetal Bovine Serum (FBS), 4mM L-glutamine, 2 mM sodium sulfate and 1% penicillin/streptomycin (Invitrogen GibcoBRL, Carlsbad, Calif., USA) at 37 ℃ and 5% CO₂Culture under ambient conditions 2 × 10⁴Cells were seeded on 96-well black plates and 24 hours later, the medium was replaced with DMEM medium containing 5.6 or 30 mM glucose and adenine was added at the indicated concentration. After 24 hours of treatment, intracellular reactive oxygen species were detected as H2 DCF-DA. After washing the cells 1 time with PBS buffer, the cells were cultured at 37 ℃ in 100. mu.M DCF for 30 minutes. DCF fluorescence was analyzed by a disc fluorescence analyzer (excitation wavelength: 485 nm; scattering wavelength: 530 nm).

The effect of adenine on reactive oxygen species generation is summarized in Table 6. Adenine significantly reduced high glucose-induced reactive oxygen species generation and was dose dependent.

Watch (6)

Adenine (microM)	Glucose (mM)	Reactive oxygen species formation (% 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

Example 11

Cancer cell growth inhibition assay

Human liver cancer cell Hep G2, human breast cancer cell MCF7 and human colon cancer cell HT29 are used for affecting the growth of cancer cells by adenine. The cells were treated with Dulbecco's Modified Eagle's Medium (DMEM) containing 10% Fetal Bovine Serum (FBS), 4mM L-glutamine, 2 mM sodium pyroltate and 1% penicillin/streptomycin (Invitrogen GibcoBRL, Carlsbad, Calif., USA) at 37 ℃ in 5% CO₂Cultivation under Environment 1 × 10⁵Cells were seeded in 6-well plates and 24 hours later, cells were treated with adenine at the indicated concentration for 72 hours and the number of surviving cells was counted. Cells were detached with trypsin-EDTA and stained with trypan blue, and viable cell number was counted with a hemocytometer.

The 50% growth inhibitory concentrations of adenine to Hep G2, MCF7, HT29 were 544.1, 537.5 and 531.9. mu.M, respectively.

Example 12

Tumor growth assay

Human hepatoma cells Hep G2 were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% Fetal Bovine Serum (FBS), 4mM L-glutamine, 2 mM sodium sulfate and 1% penicilin/streptomycin (Invitrogen GibcoBRL, Carlsbad, CA, USA) at 37 ℃ and 5% CO₂Culture under ambient conditions 5 × 10⁶Cells were injected subcutaneously into 8-week-old NOD-SCID mice. After transplantation, mice were given 5, 20, 50 mg/kg body weight adenine daily by intraperitoneal injection, and tumor size was measured every 3 days. Adenine administration significantly delayed tumor growth compared to control mice at 14 days post-transplantation.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Claims

1. Use of adenine and/or a pharmaceutically acceptable salt thereof in the preparation of a medicament for activating AMPK by direct administration at a wound to inhibit scar formation, the medicament inhibiting the growth of fibroblasts.