CN104582730A

CN104582730A - Method for improving metabolic function of mitochondria and use thereof

Info

Publication number: CN104582730A
Application number: CN201480002059.2A
Authority: CN
Inventors: 曾嘉
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-05-17
Filing date: 2014-01-23
Publication date: 2015-04-29
Anticipated expiration: 2034-01-23
Also published as: WO2014183483A1; CN104162163A; CN104582730B

Abstract

Disclosed are a method for improving metabolic function of mitochondria and use thereof, the method comprising: by inhibiting the in vivo activity of fatty acyl coenzyme A oxidase, activating the adenosine monophosphate activating protein kinase, and promoting mitochondria adipose acidification and mitochondria regeneration in target organs or tissue cells, thereby improving the metabolic function of mitochondria, strengthening insulin sensitivity in the body, and slowing cell ageing. Also disclosed is the use of fatty acyl coenzyme A oxidase inhibitors or fatty acyl coenzyme A oxidase inhibitor precursors in the preparation of drugs for treating diseases related to mitochondrial metabolism dysfunction, such as drugs for diabetes.

Description

Method for improving metabolic function of mitochondria and use thereof

A kind of method for improving Metabolism of Mitochondria function and application

Technical field

The invention belongs to biochemistry and pharmaceutical field, specifically, the present invention relates to by suppressing internal acyl coenzyme A oxidase active, promote mitochondrial fatty acid oxidase and Mitochondrial regeneration, the method for improving Metabolism of Mitochondria function.The purposes in being used to treat the medicine of Metabolism of Mitochondria dysfunction relevant disease such as diabetes is being prepared the invention further relates to acyl coenzyme A oxidase inhibitor or acyl coenzyme A oxidase inhibitor precursor.Background technology

Mitochondria (mitochondria) is a kind of semi-autonomous organelle with double membrane structure, itself there is independent genetic system.Mitochondria is the final place of aliphatic acid, carbohydrate and amino-acid oxidase, pass through the free energy discharged in oxidative phosphorylation process, it is atriphos (ATP) by adenosine diphosphate (ADP) (ADP) and phposphate, DIRECT ENERGY is provided for cell activities, is the main path that cell obtains energy.In addition, mitochondria is the major source that intracellular reactive oxygen radical (reactive oxygen species, ROS) is produced, and regulating cell apoptosis one of important pivot.

Mitochondria is one of intracellular organelle for being easiest to be damaged, when mitochondria quantity is reduced, mitochondrial DNA is undergone mutation or lacked, or respiratory chain is suppressed and causes Mitochondria abnormal, and fatty acid metabolism enzymatic activity reduction etc. reason can cause mitochondrial ATP synthesis reduction, Metabolism of Mitochondria ability is caused to decline, ROS is produced and dramatically increased, and causes mitochondria dysfunction (mitochondrial dysfunction).Mitochondria dysfunction influences the whole normal physiological function of cell, causes the generation of disease（Nunnar J and Suomalainen A., Cell, 2012, 148, 1 145-1 159).

Metabolism of Mitochondria dysfunction is the immediate cause for causing insulin resistance.Mitochondrial fatty acid oxidase activity decrease will cause triglycerides in the insulin target tissues such as liver and muscle（Triacylglycerol, TG), diglyceride diacylgly_CeRol,) etc. DG lipid is largely accumulated, wherein DG is used as signaling molecule, activate the kinases such as PKCe/ ε, and then phosphorylated insulin receptor substrate-l (IRS-l) and inactivate it, and reduce PI3 kinases (PI3K) and AKT enzymatic activitys, glucose transporter 4 (GLUT4) is caused to be reduced to indexing on film, cell is remarkably decreased to glucose intake, form insulin resistance (Lowell BB. and Shulman GL, Science, 2005,307,384-387; Erion DM. and Shulman GL, Nat. Med., 2010, 16, 400-402; Samuel VT., et. al" Lancet, 2010, 375, 2267-2277).On the other hand, Metabolism of Mitochondria function, which declines, causes the intracellular a large amount of ROS of accumulation, activate a series of protein kinases such as protein kinase C (PKC0/s), p38-MAPK, c-jun N terminal kinase (JNK), the signal proteins such as S6 kinasel (S6Kl), cause IRS IRS-1 inactivating phosphorylations（Boura-Halfon S. and Zick Y., Am. J. Physiol. Endocrinol. Metab., 2009, 296, E581-E591), then downstream signaling proteins such as phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB/Akt) isoreactivity is caused to decline, GLUT4 (GLUT4) in muscle cell endochylema is caused to be reduced to cell membrane indexing, liver hepatic glycogen synzyme (glycogen synthase) activity is significantly reduced, it is further exacerbated by internal insulin resistance (Houstis N " et. al " Nature, 2006, 440, 944-948;Styktal J. et. al., Free Radic. Biol. Med., 2012,52,46-58；Rains JL and Jain SK., Free Radic. Biol. Med., 201 1,50,567-575).In addition, beta Cell of islet mitochondria dysfunction causes islet beta-cell apoptosis and necrosis, the normal secretions (Trends Endocrinol. Metab. 2012,23,477-487) of insulin are had a strong impact on.

Insulin resistance is the common pathological characters of the metabolic diseases such as diabetes B, obesity, non-alcoholic fatty liver disease, hypertriglyceridemia.Promote insulin target organ or tissue such as liver, muscle and beta Cell of islet intracellular mitochondrial fatty acid oxidation, promote Mitochondrial regeneration, improve Metabolism of Mitochondria function, reduce these organ or tissue's inner lipids and ROS accumulation, internal insulin resistance can be significantly improved, it is important channel (the Tseng YH. for treating the metabolic diseases such as diabetes B, obesity and non-alcoholic fatty liver disease, el. al., Nat. Rev. Drug Discov. 2010,9,465-482; Serra D., et. al" Antioxid. Redox Signal., 2013,19,269-284).

Mitochondrial function regression is to cause cell ageing, causes the key link of nerve degenerative diseases, is also aging and the important pathological hallmark of nerve degenerative diseases（Beal MF., Ann. Neurol., 2005, 58, 495-505; Lin MT. and Beal MF., Nature, 2006, 443, 787-795; Karbowski M and Neutzner A., Acta Neuropathol., 2012, 123, 157-171).Due to mitochondrial DNA defect, oxidative phosphorylation occurs abnormal, cause mitochondrial obstacle, produce and accumulate a large amount of ROS, mitochondria own metabolism function is further damaged, more ROS are generated, damage is caused to nucleus DNA, activation and cell ageing coherent signal albumen, trigger cell senescence.Mitochondria has turned into the novel targets of delaying cell aging and treatment nerve degenerative diseases, improve Metabolism of Mitochondria function, it is effective means (the Reddy PH and Reddy TP. of anti-aging and treatment nerve degenerative diseases, Curr. Alzheimer Res., 2011,8,393-409;Guarente L., Cell, 2008,132,171-176).

AMP activated protein kinase (AMP-activated protein kinase, AMPK) is a kind of heterotrimer albumen, by α, three subunit compositions of beta, gamma, in liver, skeletal muscle, heart and kidney and other organs or tissue have great expression.Α Μ Ρ Κ participate in a variety of metabolic processes in organism as a kind of important protein kinase, it is referred to as cell " energy receptor ", when cell by it is any cause Α Τ Ρ generation reduce with consume it is increased stress signal when, Α Μ Ρ/Α Τ Ρ ratios increase, Thrl72 site phosphorylations in Α Μ Ρ Κ α subunits, so as to activate AMPK, regulation intracellular energy metabolism.Activation AMPK is the important means for improving Metabolism of Mitochondria function.AMPK can pass through phosphorylation acetyl-CoA carboxylase (Acetyl-CoA carboxylase; ACC) Ser79 sites suppress ACC activity; improve carnitine palmitoyltransferase l (Carnitine palmitoyltransferase 1; CPT1 it is) active; promote the sour beta-oxidation of mitochondrial fatty, suppress aliphatic acid synthesis；Mitochondria generation transcription factor PGC-la transcriptional activity can be significantly improved by activating AMPK simultaneously, promote Mitochondrial regeneration (Biogenesis).Metformin, resveratrol, the micromolecular compounds such as dinitrophenol can activate Α Μ Ρ Κ, skeletal muscle and liver Α Μ Ρ Κ can be also activated additionally by limitation energy intake (Caloric restriction) and frequent take exercise, promotes mitochondrial（Zhang BB., et. al., Cell Metab., 2009, 9, 407-416; Steinberg GR and Kemp BE., Physiol. Rev., 2009, 89, 1025-1078).By activating AMPK, Metabolism of Mitochondria function is improved, is the important channel for strengthening body insulin sensitivity and treating metabolic disease.In addition, by activating AMPK, mammal rapamycin target protein can be suppressed（MTOR it is) active, and then regulate and control the protein actives such as downstream ribosome S 6 protein kinase (ribosomal S6 kinase) and translational repressor 4EBP, delay cell senescence, it is important channel (Gwinn DM et al " the Mol. Cell; 2008; 30,214-226 for extending increased longevity and treating diseases associated with senescence; Johnson SC., et al" Nature, 2013, 493, 338-345; Laplante M and Sabatini DM., Cell, 149, 274-293; Sshin E and Depinho RA., Nat. Rev. Mol. Cell Biol, 2012, 13, 397-404; Selman C, et al" Science, 2009, 326, 140-144; Kapahi P., et al, Cell Metab., 2010, 11, 453-465)。

Peroxisome (Peroxisome) is a kind of intracellular important organelle with single-layer membrane structure, its major function is catalysis long-chain and very-long-chain fatty acid oxidation, generation short chain fatty acids return again to mitochondria and complete whole oxidizing process, and active oxygen radical (ROS) etc. in scavenger-cell.Contain a large amount of oxidizing ferment, catalysis substrate reactions generation hydrogen peroxide (H in peroxisome₂0₂), then by catalase (Catalase) by 0₂Resolve into water and oxygen.Peroxisome is very sensitive to external signal, easily occurs propagation (Proliferation), high fat diet, for a long time empty stomach, lipid-lowering medicine such as fibrate and some herbicides and agrochemical can induce peroxisome proliferation（Reddy JK. and Hashimoto T., Annu. Rev. Nutr., 2001,21,193-230).In addition, under the pathological state of mitochondria metabolic dysfunction, compensatory propagation generally occurs for peroxisome.Peroxisome proliferation is regulated and controled by peroxisome proliferation acceptor a (peroxisome proliferator-activated receptor a, PPARa). H₂0₂As signaling molecule, induction in the cell participates in the PEX serial geneses expression of peroxisome generation, the regeneration of regulation and control peroxisome (Lopez-Huertas E., et al., EMBO J., 2000,19,6770-6777) o

Acyl coenzyme A oxidizing ferment (acyl-CoA oxidase, AOX, EC 1.3.3.6), it is the enzyme of catalysis first step reaction during the sour beta-oxidation of peroxisomal fatty, it is also the rate-limiting enzyme of the sour beta oxidation process of peroxisomal fatty, molecular weight about 72 kDa, per molecule AOX contain the fast cry of certain animals dinucleotides (Flavin adenine dinucleotide, FAD) of a molecule flavine gland.AOX is catalyzed the 2-3 dehydrogenations of acyl coenzyme A carbochain, forms 2- enoyl coenzyme As (2-en₀yl-C₀A), the hydrogen atom taken off directly is combined with molecular oxygen, forms H₂0₂The expression of gene in the cell is regulated and controled by PPARa.

, in any report for improving Metabolism of Mitochondria function, also it is used for any report of the purposes for the medicine for treating Metabolism of Mitochondria dysfunction relevant disease such as diabetes as preparation without acyl coenzyme A oxidase inhibitor or acyl coenzyme A oxidase inhibitor precursor currently without on acyl coenzyme A oxidase inhibitor or acyl coenzyme A oxidase inhibitor precursor applications.The content of the invention

It is an object of the invention to provide a kind of method for improving Metabolism of Mitochondria function, acyl coenzyme A oxidase inhibitor is as the purposes of Metabolism of Mitochondria function accelerator, and acyl coenzyme A oxidase inhibitor or acyl coenzyme A oxidase inhibitor precursor are preparing the purposes in being used to treat the composition because of Metabolism of Mitochondria dysfunction associated diseases.

In the first aspect of the present invention there is provided the purposes of acyl coenzyme A oxidase inhibitor, mitochondrial fatty acid oxidase and Mitochondrial regeneration are promoted for preparing, Metabolism of Mitochondria function is improved, suppresses the composition of peroxisome proliferation；The international zymetology classifying and numbering of the acyl coenzyme A oxidizing ferment is EC 1.3.3.6.

In a preference, described target organ or tissue is the organ or tissue for expressing acyl coenzyme A oxidizing ferment；It is preferred that including：Liver, skeletal muscle, adipose tissue, heart, kidney.

In another preference, described composition is additionally operable to prevent, improve or treat Metabolism of Mitochondria dysfunction relevant disease.It is preferred that described Metabolism of Mitochondria dysfunction relevant disease includes but is not limited to：Metabolic syndrome, aging or nerve degenerative diseases；It is preferred that described metabolic syndrome includes：Insulin resistance, diabetes, obesity, Non-alcoholic fatty liver disease, hypertriglyceridemia；It is preferred that described nerve degenerative diseases include：Alzheimer's disease or parkinsonism；It is preferred that described diabetes include diabetes B, (treated with insulin resistance type 1 diabetes with insulin combination).

In a preference, the acyl coenzyme A oxidase inhibitor reduces target organ or tissue internal oxidition stress level by suppressing acyl coenzyme A oxidizing ferment, reduce insulin action target tissue internal oxidition free radical (ROS) content, improve insulin resistance, so as to prevent, improve or treat Metabolism of Mitochondria dysfunction relevant disease.

In another preference, the type 1 diabetes are characterized as that internal insulin secretion definitely lacks with insulin resistance, and blood glucose can not effectively be controlled by giving after insulin therapy；The diabetes B is characterized as that insulin resistance lacks with insulin is relative, or hypoinsulinism is with insulin resistance.

In another preference, described acyl coenzyme A oxidase inhibitor is suppression acyl coenzyme A oxidase active or the material of expression.

In another preference, described acyl coenzyme A oxidase inhibitor includes but is not limited to：Small organic molecule, small molecule inorganic matter, anti-acyl coenzyme A aoxidizes enzyme antibody, can be specifically bound with acyl coenzyme A oxidizing ferment and suppress its active nucleic acid fragment and polypeptide.

In another preference, described acyl coenzyme A oxidase inhibitor also includes inhibitor precursor.

In another preference, described acyl coenzyme A oxidase inhibitor or inhibitor precursor include but is not limited to：Carbon diacetylenic acid (the Pentacosadiynoic acid of 10,12- 25;CAS is encoded：66990-32-7, LH-919) or the carbon diine acyl coenzyme A (10,12-Pentacosadiynoyl-CoA, LH-919-CoA) of 10,12- 25.

In another preference, described composition is additionally operable to：

Suppress internal target organ or tissue acyl coenzyme A oxidase active；

Suppress internal target organ or tissue peroxisome proliferation；

Activate internal target organ or tissue AMP activated protein kinase；

Reduce internal target organ or tissue internal oxidition stress level；Or

Reduce internal insulin action target organ or tissue internal oxidition free radical (ROS) content；

Improve internal target organ or histocyte mitochondrial oxidative phosphorylation level；

Increase internal target organ or histocyte PGC-la gene expressions；

Suppress internal target organ or tissue mammal rapamycin target protein or ribosome S 6 protein kinase activity.

In another aspect of this invention there is provided a kind of composition for being used to preventing, improve or treating Metabolism of Mitochondria dysfunction relevant disease, the composition includes：Acyl coenzyme A oxidase inhibitor（It is preferred that being the inhibitor of therapeutically effective amount)；And pharmaceutically acceptable carrier or excipient.

In a preference, described acyl coenzyme A oxidase inhibitor is suppression acyl coenzyme A oxidase active or the material of expression.

In another preference, described acyl coenzyme A oxidase inhibitor includes but is not limited to：Anti- acyl coenzyme A aoxidizes enzyme antibody, can be specifically bound with acyl coenzyme A oxidizing ferment and suppress its active nucleic acid fragment and polypeptide, small organic molecule, small molecule inorganic matter. In another preference, described composition includes：Pharmaceutical composition, dietary supplement, Halth-care composition.In another preference, described acyl coenzyme A oxidase inhibitor also includes inhibitor precursor；It is preferably comprised

(but be not limited to）：The carbon diacetylenic acids of 10,12- 25（) or the carbon diine acyl coenzyme A of 10,12- 25 LH-919

(LH-919-CoA)。

In another aspect of this invention there is provided a kind of method for the composition for preparing prevention, improvement or treatment Metabolism of Mitochondria dysfunction relevant disease, methods described includes：Acyl coenzyme A oxidase inhibitor is mixed with pharmaceutically acceptable carrier or excipient, described pharmaceutical composition is obtained.

In another aspect of this invention there is provided a kind of method for preventing, improving or treating Metabolism of Mitochondria dysfunction relevant disease, methods described includes：Suppress to need the acyl coenzyme A oxidase active in patient's body for preventing, improve or treating or expression.

In a preference, described suppression acyl coenzyme A oxidase active or the method for expression are：The acyl coenzyme A oxidase inhibitor or acyl coenzyme A oxidase inhibitor precursor of effective dose are applied to the patient for needing to prevent, improve or treat.

In another preference, the target organ of the acyl coenzyme A oxidase inhibitor effect or the organ-tissue for being organized as expression acyl coenzyme A oxidizing ferment in mammal or human body, including liver organization, musculature, adipose tissue, beta Cell of islet and renal tissue etc..

In another preference, the acylcoenzyme Α oxidase inhibitors are LH-919-CoA or its precursor LH-919.Preferably selection LH-919 is implemented, and its application dosage is 0.1-3000 g/kg/d, and preferable dosage is that (g/kg/d, more preferably dosage are that (g/kg/d, more preferably dosage are 10-100 (g/kg/d to l-100 to 0.1-100.

The other side of the present invention, due to this disclosure, is obvious to those skilled in the art.Brief description of the drawings

Fig. 1 AOX inhibitor improves Metabolism of Mitochondria function in the cell, suppresses the action principle of peroxisome proliferation.N=nucleus (nuclei);M=mitochondria (mitochondria);P=peroxisome (peroxisome);MFAO=mitochondrial fatty acid oxidase（mitochondrial fatty acid oxidation)；MB=Mitochondrial regeneration（mitochondria biogenesis);PB=peroxisome regeneration (peroxisome biogenesis).

Fig. 2 LH-919-CoA suppresses the AOX activity analysis of purifying.AOX is dissolved in 20mM PBS buffer solutions (pH7.4), concentration 80nM, and 80 η Μ (Τ), the η Μ () of 240 η Μ (Δ) 480,960 η Μ (mouths are separately added into enzyme solutions）LH-919-CoA, and 800nM (o) LH-919 free acids, control group add same volume distilled water (）, in being incubated respectively after different time under 25 °C, determine AOX residual enzymic activities.

Fig. 3 LH-919-CoA suppresses AOX Determination of Kinetic Parameters.

Fig. 4 LH-919-CoA suppresses rat liver peroxisome AOX activity analysis in vitro.

Fig. 5 various concentrations LH-919-CoA (concentration is shown in abscissa) suppress rat liver peroxisome AOX activity analysis in vitro.

Fig. 6 AOX inhibitor precursors LH-919 (concentration is shown in abscissa) suppresses C57BL mouse liver AOX activity in vivo Analysis.

Scheme AOX inhibitor precursors LH-919 (concentration is shown in abscissa) and suppress Wistar rat liver AOX activity analysis in vivo.

Influence of Fig. 8 AOX inhibitor precursors to Wistar rat livers fatty acid oxidation activity.Every group of sample number n=6.Influence of Fig. 9 AOX inhibitor precursors to Wistar rat livers and skeletal muscle AOX activity.Every group of sample number n=8.

Influence of Figure 10 AOX inhibitor precursors to Wistar rat liver peroxisomal fatties acid oxidase activity.Every group of sample number n=8.

Influence of the AOX inhibitor precursors of Fig. 1 1. to Wistar Skeletal Muscle pigment c oxidase actives.Every group of sample number n=8.

Influence of Figure 12 AOX inhibitor precursors to Wistar rat livers and skeletal muscle citrate synthase activities.Every group of sample number n=8.

Influence of Figure 13 AOX inhibitor precursors to Wistar rat skeletal muscle mitochondria DNA copy numbers.Every group of sample number n

The influence that Figure 14 AOX inhibitor precursors are expressed Wistar rat skeletal muscle PGC-la mRNA.Every group of sample number n=6.

Figure 15 Western blot detect AOX inhibitor precursors to Wistar rat liver AMPKCThrl72) influence of phosphorylation level.Every group of sample number n=6.

Figure 16 Western blot detect influence of the AOX inhibitor precursors to Wistar rat livers ACC (Ser79) phosphorylation level.Every group of sample number n=6.

Figure 17 Western blot detect AOX inhibitor precursors to Wistar rat skeletal muscles AMPKCThrl 72) influence of phosphorylation level.Every group of sample number n=6.

Figure 18 Western blot detect influence of the AOX inhibitor precursors to Wistar rat skeletal muscles ACC (Ser79) phosphorylation level.Every group of sample number n=6.

Figure 19 Western blot detect AOX inhibitor precursors to Wistar rat skeletal muscle p70S6K protein expressions and p70S6KCThr389) influence of phosphorylation level (p-p70S6K).Every group of sample number n=6.

The influence that Figure 20 AOX inhibitor precursors are expressed Wistar rat skeletal muscle PEJH mRNA.Every group of sample number n

The influence of Figure 21 AOX inhibitor precursors rat liver AOX activity drug-induced to CPIB.Every group of sample number n

Influence of Figure 22 AOX inhibitor precursors to the drug-induced Skeletal Muscle pigment c oxidase actives of CPIB.Every group of sample number n=7.

Influence of Figure 23 AOX inhibitor precursors to the drug-induced rat liver citrate synthase activities of CPIB.Every group of sample number n=7.

Influence of Figure 24 AOX inhibitor precursors to the drug-induced rat skeletal muscle citrate synthase activities of CPIB.Often Group sample number n=7.

Influence of Figure 25 AOX inhibitor precursors to the drug-induced rat liver mitochondria DNA copy numbers of CPIB.Every group of sample number n=7.

Figure 26 Western blot detect the influence of AOX inhibitor precursors rat liver Α Μ Ρ Κ drug-induced to CPIB (η 172 of Τ 1) phosphorylation level.Every group of sample number η=6.

Figure 27 Western blot detect the influence of AOX inhibitor precursors rat liver ACC (Ser79) phosphorylation level drug-induced to CPIB.Every group of sample number n=6.

The influence of Figure 28 AOX inhibitor precursors rat liver mitochondria PGC- mRNA expression drug-induced to CPIB.Every group of sample number n=7.

The influence of Figure 29 AOX inhibitor precursors rat liver PEX1 mRNA expression drug-induced to CPIB.Every group of sample number n=7.

Figure 30 AOX inhibitor precursors treat the change to high fat diet inducing obesity rat liver tissue.

The treatment of Figure 31 A. AOX inhibitor precursors is resistant to the influence of (OGTT) to alloxan induced diabetic rats oral glucose.

The alloxan induced diabetic rats oral glucose tolerance offline area of blood glucose after the treatment of Figure 31 B. AOX inhibitor precursors

(AUC) compare.

Figure 32 AOX inhibitor precursors treat the influence to alloxan induced diabetic rats serum triglyceride.Figure 33 AOX inhibitor precursors treat the influence to alloxan induced diabetic rats serum free fatty acid.Influence of Figure 34 AOX inhibitor precursors treatment to alloxan induced diabetic rats liver AOX activity.Figure 35 AOX inhibitor precursors treat the influence to alloxan induced diabetic rats serum MDA.

Figure 36 AOX inhibitor precursors are treated to alloxan induced diabetic rats liver 0₂The influence of content.Figure 37 AOX inhibitor precursors treat the influence to alloxan induced diabetic rats liver tg content.Figure 38 AOX inhibitor precursors treat the influence to alloxan induced diabetic rats liver MDA.

The treatment of Figure 39 A. AOX inhibitor precursors is resistant to the influence of (OGTT) to ob/ob Mouse orals sugar.

The treatment of Figure 39 B. AOX inhibitor precursors influences on the ob/ob Mouse orals sugar tolerance offline area of blood glucose (AUC).Influence of Figure 40 AOX inhibitor precursors treatment to ob/ob mouse livers AOX activity.

The treatment of Figure 41 AOX inhibitor precursors is histological to ob/ob mouse livers to be changed.

Figure 42 AOX inhibitor precursors treat the influence to ob/ob mouse livers MDA.

Influence of Figure 43 AOX inhibitor precursors treatment to db/db mouse livers AOX activity.

Figure 44 AOX inhibitor precursors treat the influence to db/db mouse livers MDA.Embodiment

The purposes in being used to preventing, alleviate or treating the composition of Metabolism of Mitochondria dysfunction relevant disease is being prepared the invention firstly discloses acyl coenzyme A oxidase inhibitor (or inhibitor precursor).Specifically, disclosed in the present invention by giving acyl coenzyme A oxidase inhibitor (or inhibitor precursor), suppress internal acyl coenzyme A oxidase active, and then Target organ or tissue mitochondrial metabolic function are significantly improved, and reduces Cellular Oxidation stress level, improves internal insulin resistance, delaying cell aging, disease caused by treatment mitochondria dysfunction.

In specific embodiments of the present invention, by taking liver or skeletal muscle as an example, the target organ acted on as AOX inhibitor, test the inhibitory action of AOX inhibitor in vitro to liver AOX, and the inhibitory action of AOX inhibitor or AOX inhibitor precursors (precursor) in vivo to liver AOX.But of particular note is that, the target organ or tissue that AOX inhibitor or AOX inhibitor precursors are acted in vivo are not limited to liver and skeletal muscle, include the organ of any expression AOX albumen in mammal or human body, such as musculature, heart, adipose tissue, beta Cell of islet, renal tissue, because the Α Ο Χ albumen of internal these Different Organs expression is same gene transcription product, consensus amino acid sequence, and with identical physiologically active.

As used herein, described LH-919 refers to the carbon diacetylenic acids of 10,12- 25（10,12-Pentacosadiynoic acid), its CAS codings： 66990-32-7.LH-919 is LH-919-CoA precursor, and LH-919 can be converted into LH-919-CoA in vivo.

As used herein, described LH-919-CoA refers to the carbon diine acyl coenzyme A (10,12-Pentacosadiynoyl-CoA) of 10,12- 25.

Specifically, the specific embodiment of the invention includes following 3 aspects content：

1. the preparation of AOX inhibitor and the inhibitory action to AOX

The present inventor is prepared for LH-919-CoA.With LH-919 acid for raw material, high-purity LH-919-CoA is prepared for, its inhibitory action to AOX is tested.The synthetic method reference literature for preparing acyl coenzyme A by free fatty implements (Li D. et. al., J. Am. Chem. Soc, 2001,121,9034-9042).

The present invention tests inhibitory action of the AOX inhibitor to AOX out of external and animal body respectively.

The present inventor has found that LH-919-CoA can significantly inhibit liver AOX activity in vitro first.Use recombination expression first and highly purified rats'liver AOX is as target protein, determine inhibitions of the LH-919-CoA to AOX, as a result LH-919-CoA can suppress AOX activity rapidly, and this inhibitory action is irreversible.Free LH-919 acid does not have inhibitory action to AOX.

Using the complete peroxisome of rat liver as measure object, test LH-919-CoA is in vitro to AOX inhibitory action in peroxisome, and as a result LH-919-CoA energy is rapid and significantly suppresses rat liver peroxisome AOX activity.

The present invention tests inhibitory action of the AOX inhibitor precursors in animal body to AOX.

It is LH-919 conversion products in vivo that the present invention determines LH-919-CoA first.In one embodiment of the invention, Wistar rat oral gavages are given by doses LH-919, put to death after fully absorbing, rapid dissection rat, win liver, hepatic peroxisomes are separated, by analyzing rat liver peroxisome inclusion, detects and there is LH-919-CoA in peroxisome.Therefore after LH-919 absorbs in vivo, LH-919-CoA is converted into liver.

In fact, free fatty is activated in the cell to be perfectly clear for the mechanism of acyl coenzyme A, by taking peroxisome as an example, there is long-chain/pole long-chain acyl coenzyme A synzyme (acyl-CoA synthetase) on peroxisomal membrane, it is acyl coenzyme A (cyl-CoA) by free fatty (NEFA) activation, enter peroxisome afterwards Enter AOX protein actives center (Reddy JK. and Hashimoto T., Annu. Rev. Nutr., 2001,21,193-230) as catalysis substrate.

In one embodiment of the invention, C57BL mouse gavage is given by LH-919, in execution after gavage 3h, rapid dissection mouse, wins liver, separates peroxisome, determine AOX enzymatic activitys, as a result compared with control group, perfusion group C57BL mouse liver AOX activity is significantly reduced, and inhibitory action intensity and perfusion dosage are proportional.

In another embodiment of the present invention, Wistar rat oral gavages are given by LH-919, in execution after gavage 3h, rapid dissection rat, wins liver, separates peroxisome, determine AOX activity, as a result compared with control group, perfusion group Wistar rat liver AOX activity is significantly reduced, and inhibitory action intensity and perfusion dosage are proportional.

Learnt by body outer suppressioning experiment, the LH-919 that dissociates is acted on AOX unrestraints.After LH-919 absorbs in vivo, it is converted into LH-919-CoA and suppresses AOX activity.

Therefore, LH-919 is AOX inhibitor LH-919-CoA precursor, and LH-919 in vivo can be quick and irreversibly suppress AOX activity by metabolite LH-919-CoA.

2. AOX inhibitor improves the principle of target organ or histocyte mitochondrial metabolism

The present inventor has found first, by suppressing internal target organ or tissue AOX activity, can increase

AMPK (Thrl72) phosphorylation level, activate Α Μ Ρ Κ, suppress ACC activity, improve CPT1 activity, promote the sour beta-oxidation of mitochondrial fatty, improve mitochondria generation transcription factor PGC-la gene expressions, promote Mitochondrial regeneration, so as to improve Metabolism of Mitochondria function, strengthen body insulin sensitivity.In addition, AOX inhibitor is in vivo by activating AMPK, mTOR and downstream target proteins such as ribosome S 6 protein kinase activity, delaying cell aging can be suppressed.

On the other hand, AOX, which is catalyzed substrate reactions, will produce a large amount of 0₂, by suppressing internal target organ or tissue AOX activity, reduce the H that AOX catalytic reactions are produced₂0₂, the expression for the serial genes for participating in peroxisome generation, reduction target organ or histocyte endoperoxide enzyme body quantity can be significantly reduced, suppresses peroxisome proliferation, and reduce ROS generation.AOX inhibitor improves Metabolism of Mitochondria function in the cell, and the action principle for suppressing peroxisome proliferation is as shown in Figure 1.

AOX inhibitor or AOX inhibitor precursors wherein the AOX inhibitor precursors are converted into AOX inhibitor in mammal or human body, and suppress AOX activity as the purposes of Metabolism of Mitochondria function accelerator.

In one embodiment of the invention, Wistar rat oral gavages are given by LH-919 continuous 6 weeks, after put to death rat, and biochemical analysis is carried out to rat blood serum and liver and skeletal muscle tissue, as a result LH-919 medicine groups weight gain is substantially reduced compared with control group, shows that AOX inhibitor has the effect for mitigating rat body weight.Medicine group blood glucose, serum insulin and serum triglyceride content are significantly reduced compared with control group.Meanwhile, serumβ-hydroxybutyrate is significantly raised compared with control group, shows that AOX inhibitor can remarkably promote rat liver fatty acid oxidation.By determining rat liver fatty acid oxidation rates, as a result show, AOX inhibitor group liver fat acid oxidation rates are significantly raised compared with control group.Cytochrome c oxidase (Cytochrome c oxidase, COX) be weigh Mitochondria level marker enzyme, AOX inhibitor group liver and Skeletal Muscle Cell pigment oxidation enzymatic activity are dramatically increased compared with control group in embodiment, show, by suppressing AOX activity, rat liver and skeletal muscle mitochondrial oxidative phosphorylation level can be improved.Meanwhile, liver and skeletal muscle AOX activity are more right Significantly reduced according to group, hepatic peroxisomes fatty acid oxidation is significantly reduced, these results indicate that by suppressing internal AOX activity i.e. peroxisomal fatty acid oxidase, internal mitochondrial fatty acid oxidase can be promoted, Mitochondria level is improved.The present invention also compares each group liver and skeletal muscle citrate synthase activity by biochemical analysis method, mtDNA copy numbers are the characteristic index of measure of cell mitochondrial quantity, this experiment determines the citrate synthase activity and mtDNA copy numbers of experiment each group respectively, as a result show, liver and skeletal muscle citrate synthase activity and mtDNA copy numbers are significantly raised compared with control group, show that AOX inhibitor can promote Mitochondrial regeneration, significantly improve rat liver and skeletal muscle mitochondrial quantity.

By to rat liver and skeletal muscle tissue albumen western blot analyses, AOX inhibitor group rat livers and skeletal muscle AMPKCThrl72) and ACCCSer79) phosphorylation level significantly improves (ρ compared with control group<0.01), show that Α Ο Χ inhibitor, by activating Α Μ Ρ Κ, suppresses ACC activity, promotes liver and skeletal muscle mitochondrial fatty acid metabolism.RT-PCR analysis results show that Α Ο Χ inhibitor group rat skeletal muscle PGC-la gene expression doses significantly raise (p compared with control group<0.01) the key gene expression for, participating in peroxisome generation is remarkably decreased compared with control group, shows that AOX inhibitor promotes mitochondria to generate in vivo, suppresses peroxisome proliferation.

In one embodiment of the invention, by suppressing AOX activity, activate target organ or tissue such as skeletal muscle Α Μ Ρ Κ, and then suppress mammal rapamycin target protein activity, reduce ribosome S 6 protein kinase (Thr389) phosphorylation level, the expression of ribosome S 6 protein kinase is reduced, so as to significantly reduce ribosome S 6 protein kinase activity, delaying cell aging.

In another embodiment of the present invention, using clofibrate (dofibrate, CPIB) drug-induced rat model, AOX inhibitor is further explained by example and suppresses peroxisome proliferation, promotes the principle of Mitochondrial regeneration.Clofibrate is a kind of conventional lipid-lowering medicine, and it promotes peroxisome proliferation by activating PPARa.Wistar rat oral gavages are given by low dosage CPIB (100mg/kg/d) and LH-919 simultaneously, control group is only given same dose CPIB gavages, continuous gavage puts to death rat after 12 days, to serum and liver organization biochemical analysis, as a result AOX inhibitor group serum TG is significantly reduced compared with control group, AOX inhibitor group serumβ-hydroxybutyrate are significantly raised compared with control group, show that inhibitor group liver fat oxidation level is raised compared with control group.AOX inhibitor group livers and skeletal muscle COX activity are dramatically increased compared with control group, show that, by suppressing AOX activity, liver and skeletal muscle mitochondrial oxidative phosphorylation level can be improved.CPIB inductions one of peroxisome proliferation be noteworthy characterized by liver weight coefficient (；Liver weight/body weight ratio) obvious reinforcement mouthful, AOX inhibitor groups rats'liver weight coefficient significantly reduces (p compared with CPIB control groups in embodiment<0.01), show that AOX inhibitor can suppress the peroxisome proliferation of CPIB inductions in vivo.

AOX inhibitor group livers and skeletal muscle citrate synthase activity and mtDNA quantity are significantly raised compared with control group, and liver PGC- gene expression doses also significantly improve (p compared with control group<0.01), show that inhibitor group liver and skeletal muscle mitochondrial quantity are dramatically increased compared with control group.

By analyzing liver organization albumen western blot, AOX inhibitor group rat livers and skeletal muscle AMPKCThrl72) and ACCCSer79) phosphorylation level significantly rises (ρ compared with control group<0.01), show that Α Ο Χ inhibitor, by activating Α Μ Ρ Κ, promotes internal mitochondrial fatty acid oxidase and mitochondria generation, improves Metabolism of Mitochondria function.

CPIB significantly improves liver AOX activity, induces peroxisome proliferation by activating PPARa.AOX inhibitor by suppressing AOX activity, reduces 0 in vivo₂Generation, significantly reduce liver serial genes table Up to level (p<0.01) peroxisome proliferation, reduction liver weight coefficient, are suppressed.

3. AOX inhibitor or AOX inhibitor precursors are preparing the purposes in being used to treat the composition of Metabolism of Mitochondria dysfunction associated diseases

The present invention is reported by suppressing internal AOX activity first, is promoted mitochondrial fatty acid oxidase and Mitochondrial regeneration, is improved the principle of Metabolism of Mitochondria function.AOX inhibitor or AOX inhibitor precursors are used as the application of the medicine for the treatment of Metabolism of Mitochondria dysfunctional disease as Metabolism of Mitochondria function accelerator.

Specifically, the present invention illustrates respectively from two application aspects illustrates.

In a first aspect, AOX inhibitor or AOX inhibitor precursors include diabetes preparing as treatment, obesity, non-alcoholic fatty liver disease etc. using insulin resistance as the medicine of the disease of pathological hallmark in application.Metabolism of Mitochondria dysfunction is the immediate cause that body produces insulin resistance, is also the important pathological characters of these metabolic diseases.It is to improve internal insulin resistance to improve Metabolism of Mitochondria function, the main path of the metabolic disease such as treatment diabetes.

Diabetes are one kind due to h and E factor interaction, insulin it is relative or definitely lack and target tissue insulin resistance caused by carbohydrate, fat and the disorderly syndrome of protein metabolism, a kind of persistent high blood sugar is characterized, chronic, generalized metabolic disease.If not being effectively treated after onset diabetes, extensive capilary and macroangiopathy are may occur in which, the pathological change for the multisystems such as nerve, uropoiesis, circulation occur.Diabetes mainly have 2 types：(1) type 1 diabetes, islet p-cell destruction typically results in insulin and definitely lacked；（2) diabetes B, also known as Non-Insulin Dependent Diabetes Mellitus, show as whole body insulin resistance and lack with insulin is relative, or hypoinsulinism is with insulin resistance.Global diabetic in 2012 accounts for more than the 65% of overall growth number up to 3.5 hundred million people, its potential maximum growth crowd in Asia.Diabetes mellitus in China patient increases 2 times for nearly 10 years, and ill total number of persons occupies first, the whole world close to 100,000,000 people.

Currently for type 1 diabetes treatment based on insulin therapy, but type 1 diabetes patient is often accompanied by insulin resistance, and blood glucose still controls unstable after insulin therapy.For diabetes B treatment, when control body weight and motion fail effectively to control blood glucose, often treated with OHA, there are two major classes often to use oral hypoglycemic drug at present, the first kind is insulin secretion stimulators, such as sulfonylureas, this kind of medicine major side effects are easily to cause hypoglycemia, and larger to liver renal toxicity；Equations of The Second Kind is insulin sensitizer, mainly there is thiazolidinediones (TZDs) medicine such as pioglitazone etc. and biguanides such as melbine etc..Thiazolidinediones early application is more, but easily causes obesity, and the apparent side effects such as the risk of carcinoma of urinary bladder are suffered from heart failure and increase, in recent years using fewer and fewer；Easily occur the adverse side effects such as nausea, diarrhoea during biguanides use.

Under the pathological state of mitochondria metabolic dysfunction, because body insulin resistance causes glucose utilization disorders, therefore tissue more provides energy using body fat oxidation, free fatty (non-esterified fatty acid in blood, NEFA) content is significantly raised, into liver, muscle, the NEFA of the tissues such as beta Cell of islet also increases therewith, these metabolic enzyme activities and the equal compensatory of aliphatic acid beta oxidation level for organizing to participate in aliphatic acid beta oxidation in peroxisome are caused significantly to raise (Horie S. et. al., J. Biochem., 1981, 90, 1691-1696；Asayama K. et al., Mol. Cell. Biochem., 1999,194,227-234).In the embodiment of the present invention, because of Metabolism of Mitochondria dysfunction Various animal patterns such as diabetes it is big/mouse liver AOX activity significantly raises compared with normal group.

The present inventor has found first, by suppressing internal target organ or tissue AOX activity, improve Metabolism of Mitochondria function, reduce these organ or tissue's internal oxidition stress levels, internal insulin resistance is significantly improved, the effect of the metabolic diseases such as unexpected treatment diabetes, obesity, non-alcoholic fatty liver disease and hypertriglyceridemia is obtained.

The purposes in being used to treat the composition of diabetes, obesity, non-alcoholic fatty liver disease and hypertriglyceridemia is being prepared present invention additionally comprises AOX inhibitor precursors, the AOX inhibitor precursors are converted into AOX inhibitor in mammal or human body, and suppress AOX activity.For example in the embodiment of the present invention, LH-919 is AOX inhibitor precursors, and LH-919 is converted into LH-919-CoA after body absorption in liver, can quickly significantly inhibit AOX activity.

It should be appreciated that the physiologically active of AOX inhibitor and AOX inhibitor precursors in mammal or human body is identical, that is, suppress target organ or tissue AOX activity.

AOX inhibitor or AOX inhibitor precursors can treat type 1 diabetes and diabetes B.

Wherein described type 1 diabetes are characterized as that internal insulin secretion definitely lacks with insulin resistance, and blood glucose can not effectively be controlled by giving after insulin therapy；The diabetes B is characterized as that insulin resistance lacks with insulin is relative, or hypoinsulinism is with insulin resistance.

Specifically, in embodiment of the present invention, the test of pesticide effectiveness has been carried out for a kind of type 1 diabetes animal model and two kinds of diabetes B animal models.

Embodiment of the present invention carries out the animal test of pesticide effectiveness by taking a kind of AOX inhibitor precursors LH-919 as an example.Conversion product LH-919-CoA is AOX inhibitor to LH-919 in vivo, quickly can significantly suppress AOX activity in vivo.

In the test of pesticide effectiveness, the effective dose of LH-919 treatments is 0.1-3000 g/kg/d, and preferable dosage is that (g/kg/d, more preferably dosage are that (g/kg/d, more preferably dosage are 10-100 (g/kg/d to l-100 to 0.1-100.

One embodiment of this invention, first against type 1 diabetes, using alloxan induced diabetic rats model, due to alloxan selective destruction beta Cell of islet, insulin is caused definitely to lack, blood glucose is drastically raised, therefore gives alloxan diabetes rats hypodermic injection low dosage protamine zine insulin, is prevented due to the too high caused DKA of blood glucose.After Cheng Mo, diabetes rat fasting blood-glucose is significantly raised and significantly raised with insulin resistance, serum triglyceride and free fatty, and liver Α Ο Χ activity and vivo oxidation stress level are significantly raised.

Give after the treatment of various dose Α Ο Χ inhibitor precursors, treatment group's blood glucose significantly reduces (ρ compared with control group<0.01), oral glucose tolerance (OGTT) ability is significantly increased, and serum triglyceride and free fatty (NEFA) content significantly reduce (ρ<0. 01).After being treated through AOX inhibitor precursors, diabetes rat liver AOX activity significantly reduces (p<0.01), vivo oxidation stress level is significantly reduced therewith, shows that Serum MDA (malondialdeyde, MDA) content significantly reduces (p<0.01), liver 0₂(p is significantly reduced with MDA contents<0.01).

Therefore, given for type 1 diabetes with Insulin Resistance Animal Model after insulin therapy, because of internal insulin resistance, it is impossible to effectively control blood glucose.After AOX inhibitor or the treatment of AOX inhibitor precursors, by suppressing internal AOX activity, it can significantly mitigate vivo oxidation stress level, improve insulin resistance, blood glucose is reduced, the sugared tolerance of body is improved, so as to treat type 1 diabetes (being treated with insulin combination).

Another 2 embodiments of the present invention, for diabetes B, from ob/ob mouse, two kinds of animal moulds of db/db mouse Type, this 2 kinds of animal models are spontaneous diabetes B animal model, with features such as insulin resistance, hyperglycaemia, hyperinsulinemia and highs fat of blood.Liver AOX activity also significantly rise simultaneously, vivo oxidation stress level is significantly raised compared with normal group.

After being treated respectively to both diabetes B animals with AOX inhibitor precursors, treatment group's blood glucose and serum insulin level significantly reduce (p compared with control group<0. 01), and insulin resistance index HOMA-IR is remarkably decreased, and oral glucose tolerance (OGTT) is significantly increased.Serum triglyceride and free fatty (NEFA) content are significantly reduced (p<0. 01).After being treated through AOX inhibitor precursors, ob/ob mouse and db/db mouse liver AOX enzymatic activitys significantly reduce (p<0. 01), so that vivo oxidation stress level is significantly reduced, shows liver 0₂Content and MDA contents significantly reduce (p<0. 01).After being treated through AOX inhibitor precursors, ob/ob mouse inhibitor group increased weights are substantially reduced compared with control group, show AOX inhibitor in vivo by improving the target organ Metabolism of Mitochondria function such as liver and skeletal muscle, with losing weight, treat the effect of obesity.Present invention firstly discovers that, by suppressing internal AOX enzymatic activitys, promote liver fat acid oxidase, suppress liver fat acid synthesis, liver oxidative stress is reduced, improves hepatic insulin resistance, ob/ob mouse are significantly reduced, liver tg content in db/db Mice Bodies.

Therefore, for diabetes B animal model, give AOX inhibitor or the treatment of AOX inhibitor precursors, suppress internal AOX activity, vivo oxidation stress level can significantly be mitigated, lost weight, blood glucose is reduced, improve the sugared tolerance of body, reduction serum and liver tg content.Therefore AOX inhibitor or AOX inhibitor precursors can treat diabetes B, obesity, non-alcoholic fatty liver disease, the metabolic disease such as hypertriglyceridemia.

Another embodiment of the present invention, is tested using high fat diet inducing obesity rat model, and this rat model has obesity, insulin resistance, high fat of blood, the pathological characters such as fatty liver.

Give after the treatment of high fat diet inducing obesity rat AOX inhibitor precursors, AOX inhibitor group increased weights are substantially reduced compared with control group, and treatment group's fasting blood-glucose and serum insulin level significantly reduce (p compared with control group<0.01), insulin resistance index HOMA-IR is remarkably decreased.Serum triglyceride content is significantly reduced (p<0.01).Inhibitor group rat liver content of triglyceride is significantly reduced (p after being treated through AOX inhibitor precursors<0.01).Therefore, AOX inhibitor is by improving internal Metabolism of Mitochondria function, can improve internal insulin resistance, treats obesity, non-alcoholic fatty liver disease, the metabolic disease such as hypertriglyceridemia.

The purposes of second aspect, AOX inhibitor or AOX inhibitor precursors in the medicine as anti-aging and treatment nerve degenerative diseases is prepared.Mitochondria is in core status in the pathogenesis of aging and nerve degenerative diseases, Metabolism of Mitochondria dysfunction is to cause cell ageing, cause the immediate cause of nerve degenerative diseases, it is also the important pathological hallmark of these diseases, the important channel that Metabolism of Mitochondria function is anti-aging and treatment nerve degenerative diseases is improved, these conclusions are all known.

One embodiment of the invention, by suppressing target organ AOX activity in Wistar rat bodies, AMPK (Thrl72) phosphorylation level can be increased, activate AMPK, suppress ACC activity, promote the sour beta-oxidation of mitochondrial fatty, improve mitochondria generation transcription factor PGC-la gene expression doses, promote Mitochondrial regeneration, so as to improve Metabolism of Mitochondria function, the efficiency that mitochondria ROS is produced is reduced, the oxidation resistance of cell, the probability that reduction mitochondrial DNA and nucleus DNA are undergone mutation is improved.On the other hand, the embodiment of the present invention is active by suppressing target organ or tissue AOX, AMPK is activated, mammal rapamycin target protein (mTOR) activity can be suppressed, and then suppresses downstream ribosome S 6 protein kinase activity, delays cell senescence, treats diseases associated with senescence.Many experiments report is confirmed, improve Metabolism of Mitochondria function, suppress mTOR or downstream ribosome S 6 protein kinase activity, animal lifespan can be extended, treat degenerative disease, although the present invention fully demonstrates AOX inhibitor delaying cell agings without animal lifespan effect experiment, the lot of experimental data that embodiments of the invention are provided is carried out, the validity of nerve degenerative diseases is treated.Therefore, AOX inhibitor can extend mammal or the life-span of people as Metabolism of Mitochondria function accelerator, treat nerve degenerative diseases.

Above-mentioned new discovery based on the present inventor, the invention provides a kind of purposes of AOX inhibitor, for promoting mitochondrial fatty acid oxidase and Mitochondrial regeneration, improve mitochondrial metabolism, suppress peroxisome proliferation, and prepared based on this purposes and include metabolic disease such as diabetes, obesity, non-alcoholic fatty liver disease, hypertriglyceridemia etc. for preventing, improve or treat, the medicine of mitochondria dysfunction relevant disease including aging, nerve degenerative diseases etc..

As used herein, described AOX inhibitor includes lower adjustment, retarding agent, antagonist, blocking agent etc..Described AOX inhibitor refers to any activity for reducing AOX albumen, the stability of reduction AOX genes or albumen, the expression for lowering AOX albumen, the material for reducing AOX albumen effective acting times or suppressing the transcription and translation of AOX genes, these materials are used equally for the present invention, as the material useful for lowering AOX, so as to for the present invention.For example, described inhibitor includes：Small organic molecule, small molecule inorganic matter, micromolecular compound, the antibody that is combined with AOX of specificity or part, it can be specifically bound with AOX and suppress its active nucleic acid fragment and polypeptide etc., it is specific to disturb siRNA molecule or GEM 132 of AOX gene expressions etc..

As the preferred embodiment of the present invention, described AOX inhibitor is compound L H-919-CoA or its precursor LH-919.Present invention additionally comprises the isomers of above-claimed cpd, racemic modification, pharmaceutically acceptable salt, hydrate.Described " pharmaceutically acceptable salt " refers to the salt of the reaction generation such as the compound and inorganic acid, Organic Acid and Base metal or alkaline-earth metal.Compound has one or more asymmetric centers.So, these compounds can exist as racemic mixture, single enantiomter, single diastereoisomer, non-enantiomer mixture, cis or trans isomers.Described " precursor " refers to after being taken with appropriate method, and the precursor of the compound is transformed into the compound being metabolized or being chemically reacted in patient body, or the salt or solution being made up of the compound.

As another optional mode of the present invention, described AOX inhibitor can be the antibody that a species specificity is combined with AOX.Described antibody can be monoclonal antibody or polyclonal antibody.AOX protein immune animals, such as rabbit can be used, mouse, rat etc. produces polyclonal antibody；A variety of adjuvants can be used for enhancing immune response, including but not limited to Freund's adjuvant etc..Similar, expressing AOX or its, there is the cell of antigenic fragment can be used to immune animal to produce antibody.Described antibody can also be monoclonal antibody, and such monoclonal antibody can be prepared using hybridoma technology（See Kohler et al., Nature 256；495,1975；Kohler et al., Eur.J.Immunol. 6：51 1,1976；Kohler et al., Eur.J.Immunol. 6：292,1976；Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).

As another optional mode of the present invention, described AOX inhibitor can be a kind of specific siRNA molecules of AOX（siRNA) .As used herein, it is described " siRNA (small interfering RNA, SiRNA a kind of short-movie section double stranded rna molecule) " is referred to, can degrade specific mRNA by target of the mRNA of homologous complementary sequence, this process is exactly RNA interference（RNA interference) process.SiRNA can be prepared into the form of double-strandednucleic acid, and it contains a positive-sense strand and an antisense strand, and this two chains only form double-strand under conditions of hybridization.One double-stranded RNA compound can be prepared by the positive-sense strand and antisense strand that are separated from each other.Therefore, for example, complementary positive-sense strand and antisense strand are chemical syntheses, and the double-stranded RNA compound of synthesis by anneal, can be produced thereafter.

Present invention also offers a kind of composition, it contains the AOX inhibitor or AOX inhibitor precursors of effective dose, and acceptable carrier pharmaceutically or in bromatology.The composition of the present invention can be used for by suppressing AOX activity, improve target organ or tissue Metabolism of Mitochondria function, improve body insulin resistance, treat the medicine of all diseases caused by Metabolism of Mitochondria dysfunction including diabetes, obesity, non-alcoholic fatty liver disease, hypertriglyceridemia, nerve degenerative diseases such as Alzheimer's disease and parkinsonism etc., aging etc..

As used herein, the composition of " pharmaceutically or in bromatology acceptable " applies to people and mammal and without adverse side effect (such as toxicity, irritate and allergy), i.e., with rational benefit/risk than material.Term " pharmaceutically or in bromatology acceptable carrier " refers to the carrier taken for Therapeutic Administration or for health products, including various excipient and diluent.

As used herein, term " composition " includes but is not limited to：Pharmaceutical composition, dietary supplement, Halth-care composition, as long as they contain the AOX inhibitor or AOX inhibitor precursors of the present invention, as prevention, improve or treat the active component of mammal and human mitochondrion metabolic dysfunction relevant disease.

In the present invention, term " containing " represents that various composition can be applied in the mixture of the present invention or composition together.Therefore, term " main by ... constitute " and " by ... constitute " included in term " containing ".

The combination of traditional Chinese medicine of the present invention go up or bromatology on acceptable carrier, including (but being not limited to)：Olive oil, salt solution, buffer solution, glucose, water, glycerine and combinations thereof.Usual pharmaceutical preparation should match with administering mode.Pharmaceutical composition is preferably aseptically manufactured.The dosage of active component is therapeutically effective amount.

The formulation of composition of the present invention can be diversified, as long as the formulation that active component can be made effectively to reach in mammal or human body is all possible.Such as it may be selected from：Tablet, capsule, powder, particle, syrup, solution, suspension or aerosol.

In case of need, pharmaceutical composition of the invention can also be with other one or more for the effective agents in combination application of metabolic syndrome.Following one or more medicines are selected from for example, can also contain in described composition：Antidiabetic medicine, fat-reducing medicament, slimming medicine, drug for hypertension or the anticoagulation medicine of other species.When two or more administered in combination, the effect being administered alone is had typically better than.For example, the antidiabetic medicine of other species is selected from：Biguanides, sulfonylurea, meglitinide hypoglycemic medicine, alpha-glucosidase restrainer, insulin sensitizer (such as Thiazolidinediones), aP2 inhibitor, DPPIV inhibitor, SGLT2 inhibitor, insulin, GLP-1 (GLP-1) or their analog.

It should be appreciated that the invention is not restricted to the testing program of specific method described herein, including use, analysis test method and reagent etc., these can all change. With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are only illustrative of the invention and is not intended to limit the scope of the invention.The experimental method of unreceipted actual conditions in the following example, generally writes, Molecular Cloning:A Laboratory guide according to normal condition such as J. Pehanorm Brookers etc., the third edition, Science Press, the condition described in 2002, or according to the condition proposed by manufacturer.Embodiment 1, the preparation of AOX inhibitor and body outer suppressioning experiment

(1) AOX inhibitor LH-919-CoA preparation

LH-919-CoA preparation method reference literature carries out (Li D. et. al., J. Am. Chem. Soc, 2001,121,9034-9042) o concrete operations：Mg (the Sigma of LH-919 20; St. Louis; MO); add 5 mL anhydrous tetrahydro furans (tetrahydroforan; THF) (Acros; Geel; Belgium in); 0.1 mmol triethylamines (triethylamine) (Sigma is added into solution under nitrogen protection; St. Louis; MO), after mixing 10 minutes, 0.1 mmol isobutyl chlorocarbonates are added dropwise into solution under 0 °C（Isobutyl chloroformate) (Acros, Geel, Belgium), l h are stirred at room temperature.Afterwards by 50mg coacetylases sodium salt (Coenzyme A) (USB; Cleveland; OH) it is dissolved in distilled water; plus l mol/L NaOH adjust pH to 8.0; it is added drop-wise under nitrogen protection in reaction solution; continue after stirring 20 minutes, 1 M hydrochloric acid is slowly added dropwise, pH value of solution is adjusted to 5.5 6.0.Vapor away organic solvent under negative pressure, the remaining aqueous solution is extracted twice the organic solvent for removing residual with ether, collect lower floor's aqueous phase, through centrifugal ultrafiltration pipe (Millipore, Billerica, MA) filtering after LH-919-CoA is isolated and purified by RPLC (RP-HPLC).RP-HPLC separation conditions：The posts of chromatographic column hypersil C 18（The mm of 250 mm x 4. 6,5 μ η ι), using gradient elution, mobile phase Α is 25mM kaliumphosphate buffers (pH 5.9), Mobile phase B is 85% (v/v) methanol aqueous solution, and elution program Mobile phase B accounts for mobile phase cumulative volume ratio and is:0-5 min 0% B, 5-24 min 0%- 100% B, the B of 24-35 min 100%, flow velocity： 0.8 mL-rnin"¹, the nm of Detection wavelength 260,30 °C of column temperature, sample size： 100 μί.LH-919-CoA appearance times collect LH-919-CoA components about in 27min, then remove solvent through vacuum freeze drying, obtain LH-919-CoA freeze-dried powders.

(2) expression and purification restructuring AOX

The present invention screens its inhibitor using recombinant rat liver AOX as target protein, it will be appreciated that restructuring AOX and natural A OX has identical physiologically active.Recombination expressions and affinity purification reference literature method of the rats'liver AOX in E.co/ are implemented（Zeng J. et. al., Protein Expression and Purification. 2004,37,472-478), it is highly purified after AOX enzymatic activitys be 1.89U/mg, purity of protein>95%.(3) LH-919-CoA is analyzed the recombinant rat liver AOX In-vitro Inhibitory Effects of purifying

Above-mentioned highly purified recombinant rat liver AOX is dissolved in 20mM PBSs (pH7.4), concentration 80nM, various concentrations LH-919-CoA (inhibitor group) is separately added into enzyme solutions again, control group adds same volume distilled water, in being incubated respectively after different time under 25 °C, inhibitor group and control group acyl coenzyme A oxidizing ferment residual activity are determined.Acyl coenzyme A oxidase active assay method is according to document (Luo Y. et al., Arch. Biochem. Biophys., 2000,384,1-8), by Shimadzu UV- 1800 it is ultraviolet/visible spectrophotometric instrument determine 263nm absorbance changes, i.e. 2- alkene fat Generation (the ε ^ η η ^ Υ η ι Μ ^ η ι of acyl coenzyme A products¹), to determine Α Ο Χ activity.Reaction system includes 50 mmol/L PBS buffer solutions, the μ Μ palmitoyl-CoA of H 7.4,30 (Sigma, St. Louis, MO), 500ng AOX, the μ of cumulative volume 500

As a result see that Fig. 2 LH-919-CoA can quickly suppress Α Ο Χ activity, and inhibitory action increases and strengthened with LH-919-CoA concentration.Free LH-919 acid does not have inhibitory action to Α Ο Χ, and LH-919 free acids must activate that AOX activity could be suppressed after acylcoenzyme Α i.e. substrate form.

LH-919-CoA is irreversible to AOX inhibitory action, and experimental procedure is as follows：AOX is dissolved in 20mM PBS buffer solutions, pH7.4, final concentration Ι Ο Ο η Μ, 2 μ Μ LH-919-CoA are added into enzyme solutions again, in being incubated 60 min under 25 °C, to ensure that enzyme is fully acted on inhibitor, then AOX and/inhibitor mixture are fitted into bag filter (MWCO 5000), place into the mM PBSs of 2L 20 (pH 7.4), dialyse 24h in 4 °C to buffer solution, period changed a dialyzate every 4 hours, and free LH-919-CoA is removed with abundant.100nM AOX are added respective volume distilled water by control group, and remaining operation is identical with inhibitor group.Inhibitor group and control group are determined into AOX enzymatic activitys respectively after dialysis, as a result it is as shown in table 1, inhibitor group is removed by dialysing after free LH-919-CoA, its AOX activity can not be recovered, control group enzymatic activity does not have significant changes, therefore, LH-919-CoA is irreversible to AOX inhibitory action.

The LH-919-CoA of table 1 suppresses AOX Before and after dialysis o expression activitiys

Control group inhibitor group

Enzymatic activity (U/mg) relative activity (%) enzymatic activity (U/mg) relative activity (%) dialysis preceding 1.93 100%

After dialysis 0.062 3.2% note：Relative activity represents the activity value that each group the is determined percent value active with control group before dialysis.Because LH-919-CoA is irreversible to AOX inhibitory action, therefore use and k_mactTo characterize inhibition dynamics, reference literature method carries out (1-133 of Li D. et. al. J. Am. Chem. Soc. 2,001 1 19 1 1).Experimental procedure is as follows：50 nM AOX are dissolved in 20mM PBSs, pH 7.4, again 0.5 μ Μ Ι μ Μ are separately added into enzyme solutions, the μ Μ Ι Ο μ Μ of 3 μ Μ 5,15 μ Μ Z-919-CoA, in incubation 0min 5 min under 25 °C, lOmin, after 15 min and 20min, each dosage group AOX activity is determined respectively, under each LH-919-CoA concentration, is taken the logarithm with relative residual activity and incubation time is mapped, linear fit is carried out using Sigmaplotl2.0 softwares, calculating obtains apparent suppression speed k under each inhibitor concentration._bs, by k under each concentration._bsTo inhibitor concentration double-reciprocal plot, as shown in figure 3, it is 810 nM, k to AOX enzyme inhibition dynamics parameter value to calculate LH-919-CoA_tIt is worth for 3.08 min "¹ ₀

(4) LH-919-CoA is analyzed rat liver peroxisome AOX In-vitro Inhibitory Effects

Rat liver peroxisome separation reference literature implements (205-210 of Small GL. et. al. Biochem. J. 1,985 227).Operating procedure is as follows：Rat fresh liver tissue 0.5g accurately is weighed, in being shredded on ice bath, 9 times of volumes is suspended in and hooks C 10%Cw/v in slurry buffer solutions) sucrose, 3mM imidazoles, 20mM PBSs, pH 7.4), on ice bath After electronic hook slurry, 4 °C of lower 5000xg are centrifuged 10 minutes, draw supernatant.Lower sediment is resuspended in a small amount of hook slurry buffer solution 5000xg centrifugations 10 minutes again, merges supernatant and is centrifuged 30 minutes in 35000xg, lower sediment is peroxisome.

Take 200 μ_§Peroxisome（With protein refractometer) (pH 7.4) is suspended in 20mM PBSs, then add into solution 5 μ Μ LH-919-CoAC inhibitor groups）, control group adds respective volume distilled water, in being incubated under 25 °C after 0 min, 2 min, 5 min, 10 min and 20 min, and inhibitor group and control group A OX activity are determined respectively.

Peroxisome AOX activity reference literatures determine (Johnson JK. et.al., Biochemstry, 1992,31,10564-10575；Luo Y. et al, Arch. Biochem. Biophys., 2000,384,1-8), using AAS, determine AOX catalysis substrate 3-indolepropionyl-CoA (IP-CoA) and aoxidize generated trans-3-indoleacryloyl-CoA (IA-CoA) amounts to reflect AOX activity, IA-CoA extinction coefficient epsilon_{367 nm}Contain 50 mmol/L PBS buffer solutions, the μ η ι ο Ι of pH 7.4,30/L IP-CoA (purity for 26.5 mM^cm^ reaction solutions>95%), 30 μ η ι ο Ι/L FAD (Sigma, St. Louis, MO), 0.5 g/L BSA (Sangon Biotech., Shanghai), 0.02% TritonX-100 (Sangon Biotech.,), and 20 (^g peroxisomes Shanghai（With protein refractometer）.Using Shimadzu UV-1800 UV/visible spectrophotometers, determine under 367nm extinction value changes, analysis condition, the peroxisome protein content needed for 1 μ η ι ο Ι Ι Α-CoA of generation per minute is 1 unit of enzyme activity (U).

As a result as shown in figure 4, can quickly suppress rat peroxisome AOX activity using rat peroxisome as object living, LH-919-CoA is surveyed.

Another experiment, takes 200 μ_§Peroxisome（With protein refractometer) it is suspended in 20mM PBSs (pH7.4), various concentrations LH-919-CoA (inhibitor group) is separately added into solution, control group adds respective volume distilled water, in after incubation 2min under 25 °C, inhibitor each group and control group A OX activity are determined respectively, as a result as shown in figure 5, LH-919-CoA suppresses peroxisome AOX activity with adding inhibitor amount correlation in vitro.Embodiment 2, AOX inhibitor precursors suppress AOX function analysis in big/Mice Body

(1) LH-919 is converted into LH-919-CoA measuring in animal body

SPF grades of Wistar rats 6, male, body weight 220-250g, after fasting 12h, 6 rats are divided into 2 groups, control group and medicine group, every group 3, control group gives olive oil gavage 0.2mL/ only, LH-919 is dissolved in respective volume olive oil by medicine group, the g/kg of gavage LH-919 100, in putting to death whole rats after 3h, liver is won in rapid dissection, is immediately placed in liquid nitrogen and is preserved.

The measure bibliography of subcellular fraction unit inclusion acyl coenzyme A implements (Melde K. et. al., Biochem. J., 1991,274,395-400) in liver cell.Operating procedure is as follows：Liver organization 0.3g accurately is weighed, in being shredded on ice bath, 9 times of volumes is suspended in and hooks in slurry buffer solution（10% sucrose, 3mM imidazoles, 20mM PBS) on ice bath after electronic hook slurry, 4 °C of 5000xg are centrifuged 10 minutes, draw supernatant.Lower floor is resuspended in a small amount of hook slurry buffer solution 5000xg centrifugations 10 minutes again, merges supernatant and is centrifuged 30 minutes in 30000xg, obtained precipitation as peroxisome.Peroxisome is suspended in in 2 mL ultra-pure waters (4 °C) first, 200 μ 5mM HC10 are then added₄Precipitating proteins, and 20nM Lignoceryl-CoA (C24:0) as with reference to sample, protein precipitation is centrifuged in 16000xg, Supernatant K₂C0₃PH to 7.0 is adjusted, centrifugation removes KC10₄Precipitation, the freeze-dried removal solvent of supernatant, remaining solid thing is dissolved in 200 μ ultra-pure waters, and control group and medicine group each sample LH-919-CoA contents are detected by RP-HPLC, is confirmed finally by mass spectrum (MS).RP-HPLC operating conditions, chromatographic column hypersil C18 posts（250 mm X 4. 6 mm, 5 μ η ι), mobile phase Α is 25mM kaliumphosphate buffers (pH 5.9), and Mobile phase B is 85% methanol aqueous solution, and using gradient elution, gradient elution program Mobile phase B accounts for mobile phase cumulative volume ratio and is：The B of 0-5 min 0% B, 5-24 min 0%-100% B, 24-35 min 100%, flow velocity： 0.8 mL-rnin"¹, the nm of Detection wavelength 260, column temperature：30 °C, sample size： 25 μL o

As a result show, detected in LH-919-CoA, control group hepatic peroxisomes in each sample hepatic peroxisomes of medicine group and do not detect LH-919-CoA.

Therefore, after LH-919 enters liver through body absorption, LH-919-CoA is converted into, into peroxisome, is interacted as substrate and AOX.

(2) AOX inhibitor precursors are to liver AOX the Inhibitory Effects in C57BL mouse body

SPF grades of C57BL mouses 16, male female each 8, body weight 20-25g, after mouse fasting 12h, 16 mouse are divided into 2 groups, control group and medicine group, every group 8, hero female half and half.Control group gives olive oil gavage 0.2mL/ only；LH-919 is dissolved in respective volume olive oil by medicine group, gavage LH-919 20-80 g/kg.In putting to death whole mouse after gavage 3h, liver is won in rapid dissection, is immediately placed in liquid nitrogen and is preserved.It is accurate to weigh liver 0.3g, hook after the isolated peroxisome of slurry, control group and medicine group each sample AOX activity are determined respectively.

Data represent with X ± SEM, otherness between statistical procedures, each group is carried out with SPSS17.0 softwares compare to use independent samples t test.Compared with control group, * P<0.05, * * P<0.01, * * * P< 0.001.

As a result it is as shown in Figure 6, AOX enzymatic activitys are significantly reduced compared with control group in its liver organization of medicine group, show that AOX inhibitor precursors LH-919 is converted into LH-919-CoA in vivo, C57BL mouse liver AOX enzymatic activitys can be significantly inhibited, it is consistent with Vitro Experimental Results.AOX inhibitor precursors LH-919 suppresses liver AOX enzymatic activitys in C57BL mouse body has good dose dependent.

(3) AOX inhibitor precursors are to liver AOX the Inhibitory Effects in Wistar rat bodies

SPF grades of Wistar rats 12, male female each 6, body weight 220-250g after Rat Fast 5h, is divided into 2 groups, control group and medicine group, every group 6, hero female half and half.Control group gives olive oil gavage 0.2mL/ only；LH-919 is dissolved in respective volume olive oil by medicine group, gavage LH-919 10-80 g/kg, and in putting to death whole rats after 3h, liver is won in rapid dissection, is immediately placed in liquid nitrogen and is preserved.It is accurate to weigh liver 0.3g, hook after the isolated peroxisome of slurry, control group and medicine group each sample AOX enzymatic activitys are determined respectively.

As a result as shown in fig. 7, AOX enzymatic activitys are significantly reduced compared with control group in its liver organization of medicine group, show that AOX inhibitor precursors LH-919 is converted into LH-919-CoA in vivo, liver AOX activity can be significantly inhibited, with external reality Test result consistent.A0X inhibitor precursors LH-919, which suppresses rat liver A0X enzymatic activitys, has good dose dependent.Embodiment 3, by suppressing internal AOX activity, activation liver and skeletal muscle AMPK, improve liver and skeletal muscle mitochondrial metabolism

(l) AOX inhibitor precursors improve Wistar rat livers and the experiment of skeletal muscle mitochondrial metabolism

SPF grades of Wistar rats 16, male, body weight 260-280g is divided into 2 groups, control group and AOX inhibitor groups, every group 8.Control group gives olive oil gavage 0.2mL/ only；LH-919 is dissolved in respective volume olive oil by AOX inhibitor group, the g/kg of gavage LH-919 50,1 times/day of gavage, continuous gavage 21 days.Every 7 days of period determined rat body weight.

In the 22nd day 5 after gavage:Fasting blood-glucose is surveyed from rat tail vein blood sampling after 00am fasting, 3h, blood sugar detection determines (OneTouch UltraVue, Johnson and Johnson, NJ., USA) using blood glucose meter and supporting test paper.After pluck eyeball and take blood, separate serum.Insulin ELISA kit (Millipore, Billerica, MA., USA) determine serum insulin, serum triglyceride content determines (the safe clinical diagnosis Co., Ltd of Beijing Northization, Beijing using triglycerides detection kit）, serumβ-hydroxybutyrate contents use kit measurement（Sigma, St. Louis, MO, USA).

All rats are then put to death, liver is won, liver wet weights are weighed.Another 1 piece of hepatic tissue, which is cut, from right lobe of liver fixed position prepares liver hook slurry, kit measurement liver tg content.Hepatic tissue cell fatty acid oxidation activity, reference literature report method is measured (Zhang D., et al., Cell Metab., 2010,11,402-411).Hepatic tissue cell peroxisomal fatty acid oxidation rates determine reference literature report method and implement COsmundsen H., et al., Biochem J., 1989,260,215-220).

Hepatocyte cracks extraction agent by CelLytic histocytes and extracts (Sigma, St. Louis. MO. USA), extract AOX activity, citrate synthetase (citrate synthase) and cytochrome c oxidizing ferment (cytochrome c oxidase) activity are determined respectively.Citrate synthase activities are determined implements (Lopez-Lluch G " et al " PNAS, 2006,103,1768-1773) with reference to Lopez-Lluch et al. report methods.Cytochrome c oxidase activity implements (Zid BM. et al., Cell, 139,149-160) with reference to Zid et al. report methods.

Rat skeletal muscle mitochondria DNA copy number (mtDNA copy number) is determined by RT-PCR, and reference literature report method implements (He L., et al., Nucleic Acids Res., 2002,30, e68).

Western blot detect rat liver or skeletal muscle AMPK, ACC and p70S6K phosphorylation level.The liver or the mg of skeletal muscle tissue 100 of liquid nitrogen cryopreservation are taken, is shredded, RIPA histiocyte lysates is added and hooks slurry, while being separately added into protease inhibitors（Complete Protease Inhibitor Cocktail) and inhibitors of phosphatases (PhosSTOP) (Roche, Mannheim, Germany), Bio-Rad DC protein assay kit determine protein concentration (Bio-Rad, Hercules, CA., USA).After SDS-PAGE protein isolates, electricity consumption transfer method is by protein delivery to PVDF films.2h is closed with 5%BSA room temperatures.Be separately added into Α Μ Ρ Κ α, phosphorylation AMPKa Thrl72), phosphorylation ACC (Ser79), p70S6K and phosphorylation p70S6K (Thr389)-anti-(Cell Signaling, Danvers, MA., USA), with β-actin Sigma, St. Louis, MO, USA) it is used as internal reference.4 °C of shaken overnights, are washed after film plus secondary antibody normal temperature Bath l h, ECL developments are incubated in vibration, and gel image analyzers determine protein band gray scale.AMPK phosphorylation levels (p-AMPK) are with phosphorylation AMPKCThr 172)/AMPK band gray level ratios represent, ACC phosphorylation levels (p-ACC) represent with phosphorylation ACC (Ser79)/p-actin band gray level ratios, p70S6K phosphorylation levels（P-p70S6K) represent that p70S6K protein expression levels are represented with p70S6K/p-actin band gray level ratios with phosphorylation p70S6K (Thr389)/p70S6K band gray level ratios.

Fluorescence real-time quantitative PCR (real-time PCR) detects liver PGC-la, PEX1 gene expression dose.Rat liver and skeletal muscle total serum IgE extract CQiagen, Valenca, CA., USA by RNeasy kits).Using the reverse transcription reagent box synthesis chains of cDNA first (Strategene, La Jo 11a, CA., USA).Fluorescence real-time quantitative PCR (7500 Fast Real-time PCR System, Applied Biosystems) is carried out using SYBRGreen fluorescent dyes.Reaction system is cDNA 80ng, the nM of sense primer 200, anti-sense primer 200nM, 2xSYBRGreen Supermix Ι Ο μ, plus pure water is to the μ of final concentration 20.- the GTGTTCCCATGTGGTCTCAT-3 ' of PEX1 sense primers 5 ' the ,-ATGTGCTCTCTTTGGCTTGG-3 ' of anti-sense primer 5 '；Bend PGC-la upstreams | thing

5 '-CAGCAAAAGCCACAAAGACG-3 ' the ,-AGTTCCAGAGAGTTCCACAC-3 ' of anti-sense primer 5 '.Internal reference is used as using 18S rRNA.RNA expression quantity is determined by Ct values, each genetic testing is repeated 3 times by 5 samples, each sample.

Data are represented with X scholar SEM, and otherness between statistical procedures, each group is carried out with the softwares of SPSS 18.0 and is compared using t inspections, is compared with control group, * P<0.05, * * P< 0.01.

After 21 days AOX inhibitor are intervened, inhibitor group rat body weight gain in weight is substantially reduced compared with control group, shows that AOX inhibitor for treating has the effect lost weight.Blood glucose and serum insulin content are remarkably decreased compared with control group, and AOX inhibitor group serum triglycerides and liver tg content are also remarkably decreased compared with control group, as shown in table 2.

The metabolism relevant parameter of each group rat of table 2

Inhibitor group serumβ-hydroxybutyrate contents significantly raise (table 2) compared with control group, show that inhibitor group liver fat oxidation level is dramatically increased compared with control group, AOX inhibitor can improve internal liver fat acid metabolic level.Enter one Step is by determining rat liver fatty acid oxidation rates, and as a result AOX inhibitor group liver fat acid oxidation rates are significantly raised compared with control group, as shown in Figure 8.Meanwhile, rat liver AOX activity is significantly reduced compared with control group（Fig. 9), hepatic peroxisomes fatty acid oxidation is significantly reduced (Figure 10), these results indicate that the raising of rat liver fatty acid oxidation activity is realized by improving mitochondrial fatty oxidation level.Therefore, by suppressing AOX activity, mitochondrial fatty acid oxidase can be promoted, rat liver fatty acid oxidation level is improved.In addition, AOX inhibitor group Skeletal Muscle Cell pigment c oxidase actives are dramatically increased compared with control group, as shown in figure 11, show, by suppressing internal AOX activity, target organ or histocyte mitochondrial oxidative phosphorylation level can be significantly improved.

By being determined to the characteristic index citrate synthase activity and mitochondria DNA copy number that characterize mitochondria quantity, as a result show, AOX inhibitor group livers and skeletal muscle citrate synthase activity are significantly higher than control group（Figure 12), inhibitor group skeletal muscle mitochondrial DNA copy number is also significantly greater than control group (Figure 13).Fluorescence real-time quantitative PCR analysis result shows, AOX inhibitor group rat skeletal muscle PGC- gene expression doses are significantly higher than control group (Figure 14), these results show, AOX inhibitor for treating can increase PGC- gene expressions, promote Mitochondrial regeneration, significantly improve rat liver and skeletal muscle mitochondrial quantity.

Analyzed by Western blot, AOX inhibitor group rat livers and skeletal muscle AMPK (Thrl72) phosphorylation levels and ACC (Ser79) phosphorylation level are all remarkably higher than control group, as shown in figures 15-18, therefore by suppressing AOX activity, AMPK can be activated, suppress ACC activity, promote mitochondrial fatty acid oxidase.

Western blot analysis results also show that the expression of AOX inhibitor group rat skeletal muscle p70S6K albumen is substantially less than control group, inhibitor group p70S6K (Th_r389) phosphorylation level is also significantly lower than control group, as shown in figure 19.These results show, by suppressing internal target organ or tissue AOX activity, activate AMPK, mammal rapamycin target protein (mTOR) activity can be suppressed, and then reduce ribosome S 6 protein kinase (Thr389) phosphorylation level, the expression of ribosome S 6 protein kinase is also significantly lowered simultaneously, so as to reduce ribosome S 6 protein kinase activity, delaying cell aging.

The expression that rat skeletal muscle participates in the gene of peroxisome generation is substantially less than control group (Figure 20), therefore by suppressing internal AOX activity, can suppress peroxisome proliferation.

The above results show, after intervening through AOX inhibitor precursors, by suppressing internal AOX activity, reduce hepatic peroxisomes fatty acid oxidation, promote mitochondrial fatty acid oxidase simultaneously, promote mitochondria bio-regeneration, significantly improve Metabolism of Mitochondria function.Therefore AOX inhibitor or AOX inhibitor precursors are Metabolism of Mitochondria function accelerator.

In addition, AOX inhibitor or AOX inhibitor precursors are alternatively arranged as mammal rapamycin target protein or ribosome S 6 protein kinase activity inhibitor.

(2) AOX inhibitor suppresses CPIB medicine rat liver peroxisome proliferations, improves Metabolism of Mitochondria Functional Experiment

SPF grades of Wistar rats 24, body weight 200-220g is divided into 3 groups, Normal group, CPIB control groups and CPIB medicine groups, every group 8.Normal group gives olive oil gavage 0.2mL/ only；CPIB is dissolved in respective volume olive oil by CPIB control groups, gives rat oral gavage CPIBCTCI, Tokyo, Japan) 100mg/kg/d_;AOX suppresses Agent group is giving CPIB gavages 100mg/kg/d simultaneously, gives the g/kg/d of LH-919 gavages 500,1 times/day of gavage, continuous gavage 12 days.

In the 13rd day 6 after gavage:Fasting blood-glucose is surveyed from rat tail vein blood sampling after 00am fasting, 3h, blood sugar detection is determined using blood glucose meter and supporting test paper.After pluck eyeball and take blood, separate serum, serum p-hydroxybutyrate (Sigma, St Louis, MO, USA) and serum triglyceride content determined respectively（The safe clinical diagnosis Co., Ltd of Beijing Northization, Beijing).

All rats are then put to death, liver is won, liver wet weights are weighed.Another 1 piece of hepatic tissue, which is cut, from right lobe of liver fixed position prepares liver hook slurry, kit measurement liver tg content.

Hepatic tissue albumen extracts (Sigma, St. Louis. MO. USA) using CelLytic histocytes cracking extraction agent, and AOX activity, citrate synthetase and cytochrome c oxidase activity are determined respectively.

Western blot methods detection liver AMPKCThrl 72) phosphorylation and ACC (Ser79) phosphorylation level, implementation is as hereinbefore.

Fluorescence real-time quantitative PCR detects rat liver PGC-la, PEX1 gene expression dose, and implementation is as hereinbefore.

Data are represented with X scholar SEM, and otherness between statistical procedures, each group is carried out with the softwares of SPSS 18.0 and is compared using t inspections.CPIB rat inhibitor groups are compared with CPIB rat control groups, * P<0.05, * * P< 0.01.

After 12 days AOX inhibitor are intervened, CPIB inhibitor group rat blood sugars and serum insulin content are remarkably decreased compared with CPIB control groups, and inhibitor group serum triglyceride and liver tg content are also remarkably decreased (table 3).CPIB medicine group serumβ-hydroxybutyrate contents significantly raise (table 3) compared with control group, show that medicine group liver fat oxidation level is dramatically increased compared with control group, the intervention of AOX inhibitor can improve liver fat acid metabolic level.Meanwhile, liver AOX activity significantly reduces (Figure 21) compared with control group, these results indicate that the raising of rat liver fatty acid oxidation activity is realized by improving mitochondrial fatty oxidation level.Therefore, by suppressing AOX activity, peroxisomal fatty oxidation level declines, meanwhile, mitochondrial fatty acid oxidase can be promoted, the overall fatty acid oxidation level of liver is improved.

In addition, CPIB inhibitor group Skeletal Muscle pigment c oxidase actives are dramatically increased compared with control group, as shown in figure 22, show, by suppressing internal AOX activity, target organ or histocyte Mitochondria level can be significantly improved.

By being determined to the characteristic index citrate synthase activity and mitochondria DNA copy number that characterize mitochondria quantity, as a result show, CPIB inhibitor group livers and skeletal muscle citrate synthase activity are significantly higher than control group (Figure 23,24), inhibitor group liver mitochondrion DNA copy number is also significantly greater than control group (Figure 25).These results indicate that the intervention of AOX inhibitor can significantly improve rat liver and skeletal muscle mitochondrial quantity.

Analyzed by Western blot, AOX inhibitor groups liver AMPKCThrl72) phosphorylation level and ACC (Ser79) phosphorylation level be significantly higher than control group, such as Figure 26, shown in 27, therefore by suppressing AOX activity, target organ such as liver or skeletal muscle AMPK can be activated, and suppresses ACC activity, promotes target organ mitochondrial fatty acid oxidase.

By to CPIB inhibitor group and CPIB control group livers RT-PCR analyses, AOX inhibitor group liver PGC-la gene expression doses are significantly higher than control group (Figure 28), therefore, by suppressing AOX activity, activate liver and skeletal muscle AMPK, PGC-7ot gene expression doses can be significantly improved, promote liver and skeletal muscle mitochondrial generation.

By the way that to CPIB inhibitor group and CPIB control group livers RT-PCR analyses, the expression for participating in the gene PEX1 of peroxisome generation is substantially less than control group (Figure 29).Therefore by suppressing AOX activity, peroxisome generation can be suppressed.

The above results show, after intervening through AOX inhibitor precursors, by suppressing internal AOX activity, reduce hepatic peroxisomes fatty acid oxidation, liver and skeletal muscle AMPK are activated simultaneously, promote mitochondrial fatty acid oxidase, promote mitochondria bio-regeneration, significantly improve Metabolism of Mitochondria function.Therefore AOX inhibitor or AOX inhibitor precursors are Metabolism of Mitochondria function accelerator.Body weight, reducing blood lipid, liver lipid lowering experiment drop in the obese rat that embodiment 4, AOX inhibitor precursors are induced high fat diet

SPF grades of Wistar rats 24, male, body weight 220-250g is divided into 3 groups, i.e. Normal group, high fat diet (High fat diet, HFD) control group and high fat diet medicine group, every group 8 by weight average.Normal group gives normal rats word material and fed (fatty calorie ratio is 12%), and high fat diet group gives high fat word material and fed (fatty calorie ratio is 58%).Normal group and high fat diet control group give olive oil gavage 0.2mL/ only；AOX inhibitor precursors LH-919 is dissolved in respective volume olive oil by high fat diet inhibitor group, gives the g/kg of LH-919 gavages 40, and 1 times/day of gavage, continuous medicine-filling 8 weeks weighs each group rat body weight in during which every 2 weeks.

57th day fasting 3h, tail vein takes blood, and blood glucose meter determines each group fasting blood-glucose.After pluck eyeball and take blood, separate serum.Insulin ELISA kit (Millipore, Billerica, MA., USA) determines serum insulin.Kit measurement serum triglyceride content. All rats are then put to death, liver is won and weighs, 1 piece of hepatic tissue is cut from right lobe of liver fixed position rapidly, is fixed with formaldehyde rapidly, be prepared into paraffin section, through hematoxylin-eosin（Hematoxylin-eosin'HE) dye, by observation by light microscope liver fat lesion situation, every group takes 4 parts of liver samples to be analyzed.It is another to cut another 1 piece of hepatic tissue preparation liver hook slurry, kit measurement liver tg content from right lobe of liver fixed position.

Data are represented with X scholar SEM, and otherness between statistical procedures, each group is carried out with the softwares of SPSS 18.0 and is compared using t inspections.Each group sample number n=8.High fat diet medicine group is compared with high fat diet control group, * P<0.05, * * P<0.01.

After 8 weeks LH-919 intervene, high fat diet inhibitor group blood glucose and serum insulin content are remarkably decreased (table 4) compared with high fat diet control group, and insulin resistance index HOMA-IR is significantly reduced compared with control group.High fat inhibitor group serum triglyceride content is remarkably decreased compared with control group, as shown in table 4.The metabolism relevant parameter of each group rat of table 4.

Non-alcoholic fatty liver disease is largely deposited as main pathological hallmark with liver tg.High fat diet inhibitor group liver liver weight coefficient and content of triglyceride are significantly reduced compared with high fat diet control group in embodiment, as shown in table 4.Observation by light microscope liver section, as a result show, high fat diet inhibitor group liver fat drop abundance is substantially less than control group, and high fat diet control group fat deposition is based on bullous fat drop, and inhibitor group Liver fatty deposition is based on vesicle fat drop, as shown in figure 30, therefore, liver tg caused by high fat diet can be substantially reduced through AOX inhibitor for treating to deposit.

After AOX inhibitor precursors LH-919 treatments, the increase of high fat diet medicine group rat body weight is remarkably decreased (table 5) compared with high fat diet control group, illustrate that AOX inhibitor has the effect lost weight, the obesity of high fat diet induction can be prevented and treated.

The increased weight of each group rat of table 5 changes with time

Group body weight evolution (g)

8 weeks 6 weeks 4 weeks 2 weeks

General food control group 55.4 ± 2.1 102.6 ± 3.3 146.1 ± 3.7 183.7 ± 4.5 The high high the above results of 58.3 ± 3.1 106.5 ± 4.7* of *, 147.9 ± 4.8** of fat inhibitor group 184.7 ± 5.4 of fat control group 69.2 ± 3.9 132.8 ± 5.7 191.5 ± 5.9 248.4 ± 7.9 show, the treatment of AOX inhibitor precursors can mitigate insulin resistance in high fat diet inducing obesity rat body, promote body fat acid metabolic, so as to lose weight, serum triglyceride is reduced, liver tg content is reduced.

Therefore, AOX inhibitor or AOX inhibitor precursors, by promoting liver and muscle fat acid metabolic, improve internal insulin resistance as Metabolism of Mitochondria function accelerator, can treat obesity and hypertriglyceridemia, and non-alcoholic fatty liver disease.Embodiment 5, AOX inhibitor precursors are hypoglycemic to alloxan induced diabetic rats, SPF grades of lipid-lowering test Wistar rats 50, body weight 180-200g, feed with normal rats word material, feed of freely intaking.Rat is adapted to after feeding one week, and 20 rats are used as normal group, remaining 30 rat modeling.30 Rat Fast 18h pneumoretroperitoneums injection alloxan (Sigma, St. Louis, MO) 200mg/kg, after 48h, every rat skin lower injection spy protamine zine insulin (Determir, Novo Nordisk, Denmark) 4-6U/ only, continuous 7d, administration time 16:00- 16:30, to maintain the physiological function that its is basic, prevent DKA.8th day morning 5:Fasting blood-glucose is surveyed from rat tail vein blood sampling after 00am fasting, 3h, blood sugar detection is determined using blood glucose meter (OneTouch UltraVue, Johnson and Johnson, New Brunswick, NJ) and supporting test paper, similarly hereinafter.It is subcutaneously injected to alloxan induced diabetic rats after the continuous 7d of protamine zine insulin, fasting blood-glucose is increased to 22-25 mmol/L, and blood glucose is tended towards stability, and insulin resistance has been formed, totally 24 Cheng Mo.

Diabetes rat is hooked by blood sugar level after Cheng Mo is divided into 3 groups, every group 8：Diabetes rat control group, gives mL/ pcs/day of olive oil gavage 0.2；Diabetes rat low dosage AOX inhibitor precursors treatment group, LH-919 is dissolved in respective volume olive oil, gives LH-919 gavage l (^g/kg/d_;Diabetes rat high dose AOX inhibitor precursors treatment group, LH-919 is dissolved in respective volume olive oil, (the ^g/kg/d of LH-919 gavages 2 is given.Normal rat control group 8 is set in addition, gives 0.2ml/ pcs/day of olive oil gavage；Normal rat medicine group 8, LH-919 is dissolved in respective volume olive oil, gives (the ^g/kg/d of LH-919 gavages 2.Diabetes rat model group and treatment group's administration are organized simultaneously continues that protamine zine insulin, continuous 13 days is subcutaneously injected.Period is respectively at d4, d8, dl2 5:Tail vein blood determines fasting blood-glucose after 00am fasting, 3h.Dl l rows OGTT is tested, and 5:00am fasting, tail vein blood determines fasting blood-glucose after 3h, based on compare (0 min), then diabetes rat each group animal gives glucose 2.5g/kg gavages, distinguish 30 min after this, 60 min, the min tail vein bloods of standing grain B 120 determine each group blood glucose, 0-120min blood glucose-time graph is drawn, and calculates Area under the curve of blood glucose AUC_Q__12Qmm。

The 13rd day 5 after drug therapy:Eyeball is plucked after 00am fasting, 3h and takes blood, serum is separated.Determine serum triglyceride content, (the safe clinical diagnosis Co., Ltd of Beijing Northization is determined using triglycerides detection kit, Beijing), using free-fat acid detection kit measure, (Bioengineering Research Institute, Nanjing are built up in Nanjing to serum free fatty acid content）.

Isolating hepatocytes peroxisome, determines each group liver cell AOX activity, is determined by substrate of IP-CoA.H in each group liver cell₂0₂Content uses 0₂Detection kit determines (Beyotime Biotech., Haimen).Serum and Using micro MDA (MDA) detection kit measure, (Bioengineering Research Institute, Nanjing are built up in Nanjing to hepatic lipid peroxidation thing content）.

Data represent with X ± SEM, carry out otherness between statistical procedures, each group with the softwares of SPSS 18.0 and compare to use independent samples t test.Compared with normal rat control group, #P<0.05, ##P<0.01, ###P<0.001.Compared with diabetes rat model group, * P<0.05, * * P<0.01, * * * P<0.001.

Diabetes rat is after AOX inhibitor precursors LH-919 treatments are given 4 days, and blood glucose is remarkably decreased before being relatively administered, and decline level remains stable always.Blood glucose declines level and dosage correlation, as shown in table 6.OGTT test result indicates that, AOX inhibitor precursors LH-919 can significantly improve alloxan induced diabetic rats oral glucose tolerance, see Figure 31 A, compared with model control group, the AUC of LH-919 high and low dose groups_Q__12QmmArea is remarkably decreased（p<, and AUC 0.01)_Q__12QmmArea declines degree and dosage correlation, as shown in figure 31b.Table 6, AOX inhibitor precursors LH-919 are to alloxan induced diabetic rats hypoglycemic effect

Otherness, which compares, between each group uses independent samples t test.Compared with diabetes rat control group, * P<0.05, * * P<0.01, *** P<0.001.After 12 days treat, diabetes rat serum triglyceride (Figure 32) and free fatty (；Figure 33) content is remarkably decreased.Liver cell peroxisome AOX activity is determined, as a result as shown in figure 34, diabetes rat control group liver AOX activity significantly rises compared with normal group, after being treated through AOX inhibitor precursors, and treatment group liver cell AOX activity is significantly reduced compared with control group.Content of MDA is remarkably decreased 0 in (Figure 35), liver cell simultaneously₂Content is remarkably decreased (Figure 36) compared with control group.

By determining each group rat liver content of triglyceride, as a result diabetes rat treatment group liver tg content is significantly reduced compared with control group, as shown in figure 37.Diabetes rat treatment group liver MDA content is also remarkably decreased compared with control group, as shown in figure 38.

The above results show, treated by AOX inhibitor precursors, improve diabetes rat liver and Muscle Mitochondria metabolism, reduce diabetes rat liver tg and ROS contents, insulin resistance in diabetes rat body can be significantly improved, blood glucose is reduced.Therefore AOX inhibitor or AOX inhibitor precursors can treat 1 with insulin resistance Patients with type Ⅰ DM (is treated) with insulin combination.Embodiment 6, AOX inhibitor precursors are hypoglycemic to ob/ob mouse, drop body weight, reducing blood lipid and liver lipid lowering tests

SPF grades of male ob/ob mouse 16, body weight 42-45 g are used as diabetes group；C57BL mouse 16, body weight 20-22g is used as normal group.Ob/ob mouse and C57BL mouse are fed with common mouse word material, feed of freely intaking.Ob/ob mouse are adapted to after feeding 1 week, fasting 3h, and tail vein takes blood, and blood glucose meter determines fasting blood-glucose, two groups, every group 8 are divided into by blood sugar level：Ob/ob control mice groups, give O. lmL olive oil gavages；AOX inhibitor for treating groups, AOX inhibitor precursors LH-919 is dissolved in respective volume olive oil, gives (the ^g/kg/d of LH-919 gavages 5.C57BL mouse is randomly divided into 2 groups, every group 8：C57BL mouse normal group, gives 0.1ml/ pcs/day of olive oil gavage；C57BL medicine groups, LH-919 is dissolved in respective volume olive oil, gives (the ^g/kg/d of LH-919 gavages 5.Each group continues gavage 16 days.

In the 4th after medicine feed, 8,12,16d fasting 3h, tail vein take blood blood glucose meter determine fasting blood-glucose.Row OGTT experiments in 13rd day, 5 after gavage:00am fasting, tail vein blood determines fasting blood-glucose after 3h, based on compare (0 min), then ob/ob mouse each group animal gives glucose 2.0g/kg gavages, difference 30 min, 60 min, and 120 min tail vein bloods measure each group blood glucose after this, 0-120min blood glucose-time graph is drawn, and calculates Area under the curve of blood glucose AUC_Q -120min °

Survey after blood glucose within 16th day, plucked eyeball and take blood, separate serum.Insulin ELISA kit (Millipore, Billerica, MA) determines serum insulin, and calculate insulin resistance index (HOMA-IR) (Mattews DR. et. al., Diabetologia, 1985,28,412-419).Kit measurement serum triglyceride, free fatty acid content.

All ob/ob mouse are then put to death, liver is won, 1 piece of hepatic tissue are cut from right lobe of liver fixed position rapidly, fixed with formaldehyde rapidly, be prepared into paraffin section, dyed by HE, observation by light microscope liver fat lesion situation, every group takes 4 parts of liver samples to be analyzed.Simultaneously another 1 piece of hepatic tissue, kit measurement liver tg content are cut from right lobe of liver same area.

Isolating hepatocytes peroxisome, determines each group liver cell AOX activity, is determined by substrate of IP-CoA.Using micro MDA detection kit measure, (Bioengineering Research Institute, Nanjing are built up in Nanjing to hepatic lipid peroxidation level）.

Data represent with X ± SEM, carry out otherness between statistical procedures, each group with the softwares of SPSS 18.0 and compare to use independent samples t test.Compared with C57BL mouse control group, # P<0.05, ## P<0.01, ### P<0.001.Compared with ob/ob control mice groups, * P<0.05, * * P<0.01, * * * P< 0.001.

Ob/ob mouse are after AOX inhibitor precursors LH-919 treatments are given 4 days, and treatment group's blood glucose is remarkably decreased compared with ob/ob control mice groups, and decline level remains stable always, as shown in table 7.OGTT test result indicates that, through AOX inhibitor precursors LH-919 treat can significantly improve ob/ob mouse oral glucose be resistant to, see Figure 39 A, compared with ob/ob control mice groups, AOX inhibitor group AUC (M_{2Q mm}Area is remarkably decreased (p<0.01), as shown in Figure 39 B.Table 7, AOX inhibitor precursors LH-919 are to ob/ob diabetic mice hypoglycemic effects

Fasting blood-glucose (mmol/L) 16 days 12 days 8 days 4 days 0 day

C57BL mouse control group 6.9 ± 0.3 7.1 ± 0.3 7.0 ± 0.2 6.9 ± 0.3 7.0 ± 0.1

± 0.3 8.6 ± 0.4** 7.8 ± 0.4*** η the y_ of 6.9 ± 0.4 6.8 ± 0.1 6.7 ± 0.3 6.6 ± 0.2 6.6 ± 0.2 ob/ob control mices group of C57BL mouse inhibitor group, 13.1 ± 0.3 13.5 ± 0.6 13.0 ± 0.5 12.9 ± 0.6 13.2 ± 0.5 ob/ob mouse inhibitor group 13.0 | 7.3 ± 0.2*** of _ Q ^^^ are after 16 days treat, ob/ob mouse medicine group serum insulin contents are remarkably decreased (table 8), and HOMA-IR is remarkably decreased compared with control group.Ob/ob mouse treatment group serum triglyceride content is remarkably decreased (table 8) compared with control group.Liver cell peroxisome AOX activity is determined, as a result as shown in figure 40, after being treated through AOX inhibitor precursors, ob/ob mouse treatment group liver AOX activity is significantly reduced compared with control group.

Ob/ob mouse treatment group liver liver weight coefficient and content of triglyceride are remarkably decreased (table 8) compared with control group.Optical microphotograph Microscopic observation ob/ob mouse each group hepatic pathology sections, as a result show, the distribution of inhibitor group ob/ob mouse livers fat drop is substantially reduced compared with control group, is distributed in fragmentary shape, as shown in figure 41, AOX inhibitor for treating can substantially reduce ob/ob mouse liver fat depositions.Inhibitor group ob/ob mouse liver MDA contents are also remarkably decreased (Figure 42) compared with ob/ob control mices group.

The metabolism relevant parameter of each group mouse of table 8.

The above results show, give the treatment of ob/ob mouse AOX inhibitor precursors, by improving liver and skeletal muscle mitochondrial metabolism, strengthen insulin sensitivity in ob/ob Mice Bodies, ob/ob mouse blood sugars can be significantly reduced, lose weight, reduction ob/ob mice serums and liver tg content.Therefore AOX inhibitor or AOX inhibitor precursors can treat diabetes B, obesity, the metabolic disease such as non-alcoholic fatty liver disease and hypertriglyceridemia.Embodiment 7, AOX inhibitor precursors are hypoglycemic to db/db mouse, reducing blood lipid and liver lipid lowering are tested

SPF grades of male db/db mouse 16, body weight 38-42g;C57BL mouse 8, body weight 20-23g is fed With common mouse word material, feed of freely intaking.Db/db mouse are adapted to after feeding 1 week, fasting 3h, and tail vein takes blood, and blood glucose meter determines fasting blood-glucose, hooked by blood sugar level and be divided into two groups, every group 8：Db/db control mice groups, give O. lmL olive oil gavages；AOX inhibitor precursors treatment group, LH-919 is dissolved in respective volume olive oil, gives (the ^g/kg/d of db/db mouse LH-919 gavages 5.C57BL mouse normal group 8 separately is set, 0.1ml/ pcs/day of olive oil gavage is given.Each group continues gavage 16 days.

In the 4th after medicine feed, 8,12,16 days fasting 3h, tail vein takes blood, and blood glucose meter determines fasting blood-glucose.Survey after blood glucose within 16th day, plucked eyeball and take blood, separate serum.Insulin ELISA kit (Millipore, Billerica, MA) determines serum insulin, and calculates insulin resistance index (HOMA-IR).Kit measurement serum triglyceride, free fatty acid content.

All db/db mouse are then put to death, liver is won, 1 piece of hepatic tissue, kit measurement liver tg content are cut from right lobe of liver same area.Prepare liver and hook slurry, isolating hepatocytes peroxisome determines each group liver cell AOX activity, determined by substrate of IP-CoA.Hepatic lipid peroxidation level determines kit detection using micro MDA (MDA), and (Bioengineering Research Institute, Nanjing are built up in Nanjing）.

Data represent with X ± SEM, carry out otherness between statistical procedures, each group with the softwares of SPSS 18.0 and compare to use independent samples t test.Compared with db/db control mice groups, * P<0.05, * * P<0.01, * * * P< 0.001.

Db/db mouse are after AOX inhibitor precursors LH-919 treatments are given, and treatment group's blood glucose is significantly reduced compared with db/db control mice groups, and blood glucose is maintained compared with plateau, as shown in table 9.Table 9, AOX inhibitor precursors are to db/db mouse hypoglycemic effects

After 16 days treat, db/db mouse treatment group's serum insulin level is remarkably decreased (table 10) compared with control group, and HOMA-IR is significantly reduced compared with control group.Db/db mouse inhibitor group serum triglyceride (table 10) content is also significantly reduced compared with control group.Liver cell peroxisome AOX activity is determined, as a result as shown in figure 43, after being treated through AOX inhibitor precursors, db/db mouse treatment group liver AOX activity is remarkably decreased compared with control group.Meanwhile, db/db mouse inhibitor group liver tg contents（Table 10) and liver MDA content (Figure 44) significantly reduced compared with db/db control mice groups.The metabolism relevant parameter of each group mouse of table 10.

C57BL control group db/db control group db/db inhibitor groups Say ± 0.48 7.95 ± 0.45** liver tgs (the μ η ι ο/g livers of 3.2 ± 0.1 5.5 ± 0.3 5.3 ± 0.2 serum triglyceride (mmol/L) of food-intake (g/d), 1.05 ± 0.05 2.01 ± 0.1 1 1.37 ± 0.10** serum insulins (ng/mL) 1.06 ± 0.1 1 13.15）8.98 ± 0.40 33.06 ± 1.44 27.33 ± 1.15** the above results show, give the treatment of db/db mouse AOX inhibitor precursors, by improving liver and skeletal muscle mitochondrial metabolism, insulin resistance in db/db Mice Bodies can be improved, significantly reduce blood glucose, reduction serum and liver tg content.Therefore, AOX inhibitor or AOX inhibitor precursors can treat diabetes B, the metabolic disease such as non-alcoholic fatty liver disease and hypertriglyceridemia as Metabolism of Mitochondria function accelerator.All documents referred in the present invention are all incorporated as reference in this application, are individually recited just as each document as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims limited range.

Claims

Standing grain

1. acyl coenzyme A oxidase inhibitor is preparing promotion mitochondrial fatty acid oxidase and Mitochondrial regeneration, the purposes in the composition of Metabolism of Mitochondria function or suppression peroxisome proliferation is improved；The international zymetology classifying and numbering of the acyl coenzyme A oxidizing ferment is EC 1.3.3.6.

2. the purposes as described in claim 1, it is characterised in that described mitochondria is the mitochondria of target organ, tissue or cell.

3. purposes as claimed in claim 2, it is characterised in that described target organ or tissue is the organ or tissue for expressing acyl coenzyme A oxidizing ferment；It is preferred that including：Liver, skeletal muscle, adipose tissue, heart, kidney.

4. the purposes as described in claim 1, it is characterised in that described composition is additionally operable to prevent, improve or treat Metabolism of Mitochondria dysfunction relevant disease.

5. purposes as claimed in claim 4, it is characterised in that described Metabolism of Mitochondria dysfunction relevant disease includes：Metabolic syndrome, aging or nerve degenerative diseases；It is preferred that described metabolic syndrome includes：Insulin resistance, diabetes, obesity, non-alcoholic fatty liver disease, hypertriglyceridemia；It is preferred that described nerve degenerative diseases include：Alzheimer's disease or parkinsonism；It is preferred that described diabetes include diabetes B, with insulin resistance type 1 diabetes.

6. purposes as claimed in claim 1, it is characterised in that described acyl coenzyme A oxidase inhibitor is suppression acyl coenzyme A oxidase active or the material of expression.

7. purposes as claimed in claim 6, it is characterised in that described acyl coenzyme A oxidase inhibitor includes：Small organic molecule, small molecule inorganic matter, anti-acyl coenzyme A aoxidizes enzyme antibody, can be specifically bound with acyl coenzyme A oxidizing ferment and suppress its active nucleic acid fragment and polypeptide.

8. purposes as claimed in claim 1, it is characterised in that described acyl coenzyme A oxidase inhibitor also includes inhibitor precursor.

9. purposes as claimed in claim 8, it is characterised in that described acyl coenzyme A oxidase inhibitor or inhibitor precursor include：10,12- bis- ten five carbon diacetylenic acids, its CAS is encoded to 66990-32-7.

10. purposes as claimed in claim 1, it is characterised in that described composition is additionally operable to：

Suppress internal target organ or tissue acyl coenzyme A oxidase active；

Suppress internal target organ or tissue peroxisome proliferation；

Activate internal target organ or tissue AMP activated protein kinase；

Reduce target organ or tissue internal oxidition stress level；

Reduce insulin action target organ or tissue internal oxidition free-radical contents；

Improve internal target organ or histocyte mitochondrial oxidative phosphorylation level；

Increase internal target organ or tissue PGC- gene expressions；

Suppress internal target organ or tissue mammal rapamycin target protein or ribosome S 6 protein kinase activity.

11. a kind of composition for being used to preventing, improve or treating Metabolism of Mitochondria dysfunction relevant disease, it is characterised in that the composition includes：

Acyl coenzyme A oxidase inhibitor；And

Pharmaceutically acceptable carrier or excipient.

12. the composition as described in claim 11, it is characterised in that described Metabolism of Mitochondria dysfunction relevant disease includes：Metabolic syndrome, aging or nerve degenerative diseases；It is preferred that described metabolic syndrome includes：Insulin resistance, diabetes, obesity, non-alcoholic fatty liver disease, hypertriglyceridemia；It is preferred that described diabetes include diabetes B, with insulin resistance type 1 diabetes；It is preferred that described nerve degenerative diseases include：Alzheimer's disease or parkinsonism.

13. the composition as described in claim 11, it is characterised in that described acyl coenzyme A oxidase inhibitor is suppression acyl coenzyme A oxidase active or the material of expression.

14. the composition as described in claim 11, it is characterised in that described acyl coenzyme A oxidase inhibitor includes：Anti- acyl coenzyme A aoxidizes enzyme antibody, can be specifically bound with acyl coenzyme A oxidizing ferment and suppress its active nucleic acid fragment and polypeptide, small organic molecule, small molecule inorganic matter.

15. the composition as described in claim 11, it is characterised in that described acyl coenzyme A oxidase inhibitor also includes inhibitor precursor；It is preferably comprised：10,12- bis- ten five carbon diacetylenic acids, its CAS is encoded to 66990-32-7.

16. a kind of prepare prevention, the method for the composition for improving or treating Metabolism of Mitochondria dysfunction relevant disease, it is characterised in that methods described includes：Acyl coenzyme A oxidase inhibitor is mixed with pharmaceutically acceptable carrier or excipient, described pharmaceutical composition is obtained.

17. a kind of method for preventing, improving or treating Metabolism of Mitochondria dysfunction relevant disease, it is characterised in that methods described includes：Suppress to need the acyl coenzyme A oxidase active in patient's body for preventing, improve or treating or expression.

18. method as claimed in claim 17, it is characterised in that described suppression acyl coenzyme A oxidase active or the method for expression are：The acyl coenzyme A oxidase inhibitor or acyl coenzyme A oxidase inhibitor precursor of effective dose are applied to the patient for needing to prevent, improve or treat.