CN109200287A

CN109200287A - Directly or indirectly application of the substance of regulation YB-1 phosphorylation in terms of the drug of preparation treatment inflammatory factor associated diseases

Info

Publication number: CN109200287A
Application number: CN201810783225.7A
Authority: CN
Inventors: 刘斌
Original assignee: Jilin Zhongtai Biological Technology Co Ltd
Current assignee: Jilin Zhongtai Biological Technology Co Ltd
Priority date: 2017-08-03
Filing date: 2018-07-17
Publication date: 2019-01-15
Anticipated expiration: 2038-07-17
Also published as: CN108144060A; CN109200287B; WO2019024518A1

Abstract

The present invention " directly or indirectly application of the substance of regulation YB-1 phosphorylation in terms of the drug of preparation treatment inflammatory factor associated diseases ", belongs to biomedicine field.The application is characterized in: the drug is using YB-1 as target spot；And the active constituent of the drug includes the substance of direct or indirect regulation YB-1 phosphorylation, and achievees the purpose that treat inflammatory factor associated diseases by the expression quantity of regulation inflammatory factor.Another aspect of the present invention also provides a kind of screening technique of drug for treating inflammatory factor associated diseases.The present invention has found the relationship of YB-1 phosphorylation inhibitor and inflammatory factor pioneeringly, and can achieve the purpose that treat inflammatory factor associated diseases by lowering the expression quantity of proinflammatory factor by experimental verification YB-1 phosphorylation inhibitor.

Description

Directly or indirectly the substance of regulation YB-1 phosphorylation is treated caused by inflammatory factor in preparation Application in terms of the drug of disease

Technical field

The invention belongs to biomedicine fields, and in particular to directly or indirectly the substance of regulation YB-1 phosphorylation is controlled in preparation Treat the application in terms of the drug of inflammatory factor associated diseases.

Background technique

The area Y binding protein -1 (Y-box-binding protein 1, YB-1) is that a kind of specific binding target gene opens A kind of transcription factor of Y-Box sequence (highly conserved cis- DNA sequence dna), belongs to cold shock inside mover and enhancer A member in albumen (Cold shock proteins) superfamily, one section of highly conserved cold shock region that it contains (Cold shock domain, CSD), is exactly the binding site of itself and nucleic acid.The albumen code name of YB-1 are as follows: Protein Symbol:P67809-YBOX1_HUMAN is made of 324 amino acid, and molecular weight is 35924 dalton.YB-1 is widely present In microorganism, plant, animals and humans, on a molecular scale, participates in DNA and repair, mRNA transcription, montage, regulating mRNA The biological processes such as stability, translation；On a cellular level, YB-1 is thin in cell Proliferation and differentiation, cellular stress, tumour It plays an important role in the conversion of born of the same parents；In terms of clinical pathology, YB-1 is proved, relevant with the generation of a variety of diseases, for example, Tumour, including liver cancer, breast cancer, adenocarcinoma of colon, lung cancer；Liver fibrosis；Inflammation；Atherosclerosis；Organ transplant rejection is anti- It answers；Reperfusion injury；YB-1 also can be used as the marker of diagnosing tumor and/or prognosis.

The inflammation of human body is a kind of defensive biological respinse to destructive stimulus, to eliminate destructive stimulus caused by body Damage (including pathogen is eliminated and removes, the tissue of the necrosis as caused by destructive stimulus and inflammation or damage is cleared up, and Start the reparation to body).Leucocyte (T cell, B cell, mast cell, thick liquid cell, granulocyte, Dendritic Cells etc.) is The main cellular of inflammatory reaction, but all cell types of human body include that blood platelet can all work in inflammatory reaction. It is mainly one group of small egg by the cytokine chemokine that leucocyte (such as macrophage, lymphocyte, fibroblast) generates The general name of leucocyte Auto-regulator, including interleukins interleukin, interferon interferon, cell stimulation factor Colony stimulating factor, chemotactic factor (CF) chemokine, lymph element lymphokine, monokaryon element monokine, And tumor necrosis factor tumor necrosis factor etc..Herein the egg to play an important role in the occurrence and development of inflammation White molecule (mainly including cytokine) is known as inflammatory factor.These factors can activate or close the cell for participating in inflammatory reaction Function, thus the process of leading inflammation occurrence and development.Although inflammation is the defensive reaction of human body, when cannot terminate in due course When with regard to occurrence injury health disease.

The inflammatory factor being concerned in cytokine is chemotactic factor (CF) and interleukins.Chemotactic factor (CF) chemokine by More than ten structures are made of the albumen that larger homology, molecular weight are mostly 8~10kD.These albumen contain one in aminoterminal more A or two cysteines.According to the arrangement mode of cysteine, chemotactic cytokine is divided into subfamily.Two and half Guangs Propylhomoserin belongs to the Asia α man by the chemotactic cytokine that Cys-X-Cys (any amino acid-cysteine of cysteine -) mode arranges Race, also referred to as CXC chemotactic cytokine；The chemotactic cytokine arranged in a manner of Cys-Cys belongs to β subfamily, and also referred to as CC becomes The property changed cell factor.The chemotactic cytokine of only one cysteine of aminoterminal claims γ subfamily chemotactic cytokine, Also referred to as C chemotactic cytokine.Chemotactic cytokine is mainly secreted by the stroma cell in leucocyte and hematopoieticmicroenviron-ment, can Any cell with Differential expression of chemokine receptors is acted on, and there is chemotactic and activation to their target cell. IL-8 is the representative of α subfamily system, has chemotactic or enrichment to neutrophil leucocyte.Monocyte chemoattractant protein-1 (monocyte chemoattractant protein-1, MCP-1 or CCL-2) is the representative of β subfamily, can chemotactic or richness Collect monocyte, basophilic granulocyte, memory t cell and Dendritic Cells.Lymphotactin (lymphotactin) be γ subfamily representative, have chemotactic or enrichment to lymphocyte.MCP-1 is primarily involved in inflammation Occurrence and development process, including atherosclerosis, systemic loupus erythematosus, rheumatic arthritis and psoriasis.Although MCP1 is anti- Body and artificial reconstructed MCP1 are used as the inhibitor of MCP1 to treat the diseases such as wound, ephritis, but exempt from due to heterologous antibody Epidemic disease source property and the inhibiting effect of artificial reconstructed MCP1 be not good enough, so that they should not be used in treatment artery sclerosis, rheumatism joint The chronic diseases such as inflammation, lupus erythematosus.Pitavastatin class lipid-lowering medicine is the clinical treatment for being widely used in artery sclerosis, such medicine Side effect includes: myositis, myalgia, myolysis, the disturbance of consciousness, tumour, hepatopathy, hemorrhagic stroke, hyperglycemia, insomnia, sexual function Lack of proper care (Am J Cardiovasc Drugs.2008；8(6):373-418).There is presently no a kind of ideal regulation MCP1 or The drug for preventing and treating artery sclerosis.Another kind of cytokine-interleukins has had found more than 50 in human body, is broadly divided into 4 Class, they play main adjustment effect to the immune function of human body.Such as interleukin-6 (IL6) therein, its high expression It can promote the occurrence and development of following disease: atherosclerosis, diabetes are depressed, senile dementia, systemic loupus erythematosus, Myeloma, prostate cancer, rheumatism, asthma and Behcet's disease Behcet ' s disease.Clinically still lack at present safe and effective Regulation IL6 drug.

In addition to cytokine, certain enzymes, enzyme inhibition albumen, receptor and hormone also adjust the pathologic process of inflammation.

So far, the mankind are still extremely limited to the treatment method of above-mentioned inflammatory factor related disease and less effective.

Summary of the invention

The present invention is based on drawbacks described above existing for this field and deficiencies, provide the disease that a kind for the treatment of is participated in by inflammatory factor The application of the drug and such drug of disease provides a kind of better drug of effect and new use to treat above-mentioned various diseases Medicine selection.

Technical scheme is as follows:

Directly or indirectly the substance of regulation YB-1 phosphorylation is in terms of the drug of preparation treatment inflammatory factor associated diseases Using.

The drug is using YB-1 as target spot；And the active constituent of the drug includes directly or indirectly regulating and controlling YB-1 phosphorylation Substance, and by regulate and control inflammatory factor expression quantity come achieve the purpose that treat inflammatory factor associated diseases.

The inflammatory factor is the inflammatory factor of YB-1 regulation；The inflammatory factor associated diseases include: inflammation, glycosuria Disease, tumour, autoimmunity disease, immune defense, fat and encephalopathy；

The inflammation is preferably atherosclerosis, asthma, rheumatic arthritis；The encephalopathy preferably be selected from dementia, epilepsy, Depression.

The active constituent of the drug includes directly or indirectly downward, and/or, directly or indirectly inhibit YB-1 phosphorylation Substance；

Further, the direct or indirect downward, and/or, directly or indirectly the substance of inhibition YB-1 phosphorylation includes YB-1 phosphorylation inhibitor；

It is further preferred that the YB-1 phosphorylation inhibitor lowers the proinflammatory factor of expression.

The YB-1 phosphorylation inhibitor is selected from by any one of following substances or appoints several groups formed: promoting YB-1 The substance of dephosphorylation, the substance for blocking or delaying YB-1 phosphorylation, downward or inhibit YB-1 protein kinase activity object Matter, up-regulation or the substance for exciting YB-1 phosphatase activity.

Opposite with the YB-1 phosphorylation inhibitor is YB-1 phosphorylation reinforcing agent；

The YB-1 phosphorylation reinforcing agent is selected from by any one of following substances or appoints several groups formed: blocking or prolongs The substance of slow YB-1 dephosphorylation, the substance of up-regulation or excitation YB-1 phosphorylation, up-regulation excite the protein kinase of YB-1 living Property substance, inhibition or lower YB-1 phosphatase activity substance；

The proinflammatory factor that the YB-1 phosphorylation inhibitor lowers expression be selected from MCP-1, Ccl2, Ccl7, Serpine1, Ecm1、Serpinf1、 Cd55、Il6、Uaca、Adora2b、Tfrc、Cx3crl、Calcrl、Chi3l1、Mecom、Pde5a、 Serping1、Il33、Ada、RT1-Db1、 Ccl12、Ciita、Ccl11、Ace、Masp1、Tril、Acp5、Ghrl。

The YB-1 phosphorylation inhibitor includes: substance shown in formula I:

Formulas I；And/or

Dexamethasone；And/or

YB-1 mutain；

The YB-1 mutain refers to, obtains after the 102 site serines mutation of the amino acid sequence of wild type YB-1 albumen The mutain arrived.

The active constituent of the drug further includes the polymer of the YB-1 phosphorylation inhibitor, and/or, the YB-1 phosphorus Acidification inhibitors pharmaceutically acceptable salt compounds；And/or ester type compound；And/or；Synergy class compound.

The drug further includes pharmaceutically acceptable auxiliary material and/or carrier.

A kind of screening technique of drug that treating inflammatory factor associated diseases, which is characterized in that using can be direct or indirect The substance for regulating and controlling YB-1 phosphorylation carries out Pathological experiment as drug candidate, and/or, clinical test, and/or, it treats, screening Provide active constituent of the substance as the drug that be effective, and/or generating curative effect.

The substance that YB-1 phosphorylation can directly or indirectly be regulated and controled includes YB-1 phosphorylation inhibitor；

The YB-1 phosphorylation inhibitor preferably is selected from: the substance of YB-1 dephosphorylation can be promoted, and/or, it blocks or delays The substance of YB-1 phosphorylation, and/or, the substance of the protein kinase activity of downward or inhibition YB-1, and/or, up-regulation or excitation The substance of YB-1 phosphatase activity.

Present invention firstly provides a kind of for treating the drug for the disease that inflammatory factor is participated in, which is characterized in that with YB- 1 is drug target；And the active constituent of the drug includes the substance that can directly or indirectly regulate and control YB-1 phosphorylation.

The present invention has found the anti-disease mechanism for the disease that said medicine treatment inflammatory factor is participated in: rat pioneeringly The transcript profile of YB1 mutant (- 100 deletion form cell line of serine, phosphorylation YB1 level decline 50% or so, see Fig. 5 C) Sequencing and its bioinformatic analysis as a result, it has been found that: the downward of YB-1 phosphorylation leads to 27 kinds of inflammatories of rat smooth muscle cell The mRNA level in-site of the factor is significantly lowered, (see Fig. 7 A).The inflammatory factor that these factors regulate and control in collectively referred to herein as YB1.These quilts The inflammatory factor of downward is in height expression with known a variety of diseases with row." downward of YB1 phosphorylation is to these inflammatory factors Whole inhibiting effect can make lower YB1 phosphorylation this substance have the function of while lowering a variety of inflammatory factors, thus With the incomparable anti-inflammatory effect of current drug ".The present invention has had proven to downward YB1 phosphorus in molecular biology verifying The substance of acidification, MK2206, can lower in the 27 kinds of inflammatory factors lowered by non-phosphorylating YB1 (3dS) except Serpinf1 and 25 kinds (Fig. 7 B) except Tfrc.Animal embodiment of the invention with MK2206 confirm lower YB1 phosphorylation to mainly by The inhibiting effect of artery sclerosis that is that MCP1 is mediated and being participated in by a variety of (such as Fig. 2) other inflammatory factors.YB1 phosphorylation Lower the mRNA level in-site downward that can be realized by lowering transcription or rna stability to the above-mentioned factor.Its work to MCP-1 With the regulation for being direct participation MCP-1mRNA stability.Under degradation of the YB-1 of non-phosphorylating by promoting MCP-1mRNA Adjust MCP-1 horizontal, to reduce monocyte in the enrichment of local organization.The YB-1 of non-phosphorylating is conducive to combine UK114 (nuclease albumen) and form a kind of compound at least containing YB-1, UK114 and GR (glucocorticoid receptor), The compound reduces intracellular monocyte chemoattractant protein-1 (monocyte chemoattractant by selectivity Protein-1:MCP-1 mRNA stability) and the expression for lowering MCP-1.Related molecular biology experiment result is shown in Fig. 4. YB-1 plays a major role to the mRNA stability of regulation MCP-1.The mildness of intracellular non-phosphorylating YB-1, which increases, (to be generated Weaken in the dephosphorylation enhancing of YB-1 or phosphorylation, or both synergistic effect) expression of MCP-1 can be significantly inhibited, And then inhibit enrichment of the monocyte in local organization, it is finally reached inhibition, is mitigated, or prevention is by using MCP-1 as representative Inflammatory factor associated diseases.Thus, regulation YB-1 phosphorylation energy Effective Regulation MCP-1 is controlled in turn to be drawn by MCP-1 imbalance The disease risen.The Atherosclerosis Model of present invention transgenic mice confirms the effect of the anti-disease mechanism.Although this hair The MCP-1 in inflammatory factor that bright zoopery only regulates and controls YB1 is verified, but by MK to artery sclerosis Inhibit to infer in 80% or more result (Fig. 2,3): MK not only inhibits MCP -1 in animal model, further suppresses other The inflammatory factor of artery sclerosis is participated in, because moving in the mouse that MCP-1 receptor CCR2 and inflammatory factor CX3CL1 are knocked out altogether The inhibiting rate of arteries and veins hardening is 66% or so.

The present invention verifies class of the present invention by taking the mainly atherosclerosis animal model caused by MCP-1 imbalance as an example The drug therapeutic effect of disease that the YB1 inflammatory factor regulated and controled is participated in.

In specific embodiment of the present invention, the MCP 1 and other inflammatory factors regulated and controled by YB1 The disease of participation includes: inflammation (atherosclerosis, arthritis, asthma etc.), patients with type Ⅰ DM, tumour, and autoimmunity disease is exempted from Epidemic disease defence, fat and encephalopathy (dull-witted, depression, epilepsy etc.).

In some embodiments of the invention, the active constituent of the drug includes directly or indirectly downward, and/or, directly Connect or inhibit indirectly the substance of YB-1 phosphorylation；

In a further embodiment, the direct or indirect downward, and/or, directly or indirectly inhibit YB-1 phosphorylation Substance include YB-1 phosphorylation inhibitor.The present invention original ground during the expression mechanisms of research regulation MCP-1 Have found that the inhibitor of effect and YB-1 phosphorylation of the YB-1 phosphorylation in regulation MCP-1 expression expresses MCP-1 Inhibiting effect.

" translation growth factor-β ", " MCP 1 ", " MCP-1 ", " Ccl2 ", " CCL- herein 2 " all refer to same substance, the meaning that those skilled in the art having the same can routinely understand.

In some specific embodiments, the YB-1 phosphorylation inhibitor is selected from by any one of following substances or appoints The group of several compositions: the substance of YB-1 dephosphorylation, the substance for blocking or delaying YB-1 phosphorylation, downward or inhibition can be promoted Substance, up-regulation or the substance for exciting YB-1 phosphatase activity of the protein kinase activity of YB-1.

Especially those highly selective directly result in YB-1 dephosphorylation or highly selective direct blocking YB-1 phosphorylation Substance and it is highly selective lower YB-1 the active substance of protein kinase (kinase) and its highly selective up-regulation YB- 1 phosphatase (phosphatase) active substance.

The effect of protein kinase is to make target protein phosphorylation, and being remarkably decreased for protein kinase can lead to protein kinase Gross activity decline, lowering or inhibiting protein kinase activity is the purpose that can reach inhibition or lower target proteins phosphorylation；

On the contrary, the effect of phosphatase is dephosphorylation (dephosphorylation), the activity of up-regulation or excitation target proteins phosphatase, The dephosphorylation of phosphatase can be made to act on to strengthen, to enhance the dephosphorylation of target proteins, and then play inhibition or Lower the purpose of target proteins phosphorylation.

The YB-1 phosphorylation reinforcing agent is selected from by any one of following substances or appoints several groups formed: blocking or prolongs The substance of slow YB-1 dephosphorylation, the substance of up-regulation or excitation YB-1 phosphorylation, up-regulation excite the protein kinase of YB-1 living Property substance, inhibition or lower YB-1 phosphatase activity substance.

Therefore, the YB-1 phosphorylation inhibitor may also is that and above-mentioned YB-1 phosphorylation reinforcing agent counterproductive Substance, and/or, YB-1 dephosphorylation reinforcing agent.

In further specific embodiment, the YB-1 phosphorylation inhibitor is that substance shown in formula I is (herein It is called MK, MK2206):

Formulas I.

In further embodiments, the YB-1 phosphorylation inhibitor is dexamethasone or YB-1 mutain；It is described YB-1 mutain refers to, the mutation egg obtained after 102 site serines of wild type YB-1 protein amino acid sequence are mutated It is white.The sequence of the wild type YB-1 albumen is as shown in SEQ ID NO.1.

In a further embodiment, the active constituent of the drug further includes the polymerization of the YB-1 phosphorylation inhibitor Object, and/or, the YB-1 phosphorylation inhibitor pharmaceutically acceptable salt compounds；And/or ester type compound；With/ Or；Synergy class compound.

In a still further embodiment, the drug further includes pharmaceutically acceptable auxiliary material and/or carrier.

Another aspect of the present invention is provided for treating MCP 1 and by other inflammation of YB1 regulation The screening technique of the drug for the disease that sex factor participates in, which is characterized in that YB-1 phosphorylation can directly or indirectly be regulated and controled by using Substance carries out Pathological experiment as drug candidate, and/or, clinical test, and/or, treatment is filtered out with effect, and/or The substance for generating curative effect is used as the active constituent of the drug.

In the specific embodiment of screening technique, the substance of the direct or indirect regulation YB-1 phosphorylation includes YB-1 Phosphorylation inhibitor；

Further, the YB-1 phosphorylation inhibitor preferably is selected from: the substance of YB-1 dephosphorylation can be promoted, and/or, resistance Substance that is disconnected or delaying YB-1 phosphorylation, and/or, the substance of the protein kinase activity of downward or inhibition YB-1, and/or, on Adjust or excite the substance of YB-1 phosphatase activity.

The present invention be also claimed can directly or indirectly regulate and control the substance of YB-1 phosphorylation prepare it is thin for treating monokaryon Born of the same parents' chemotactic protein 1 and by YB1 regulation other inflammatory factors participate in disease drug in terms of purposes, which is characterized in that will The substance that YB-1 phosphorylation can directly or indirectly be regulated and controled is placed on the commodity for indicating inflammatory factor associated diseases therapeutical uses In packing box；

Preferably, the substance that can directly or indirectly regulate and control YB-1 phosphorylation includes YB-1 phosphorylation inhibitor；

It is further preferred that the YB-1 phosphorylation inhibitor is selected from: it can promote the substance of YB-1 dephosphorylation, and/ Or, the substance of YB-1 phosphorylation is blocked or delays, and/or, the substance of the protein kinase activity of downward or inhibition YB-1, and/ Or, the substance of up-regulation or excitation YB-1 phosphorylase activities.

Under the premise of the Patent Law of some countries allows, the present invention, which is also claimed, directly or indirectly to be regulated and controled Indication of the substance of the area Y binding protein -1 (Y-box-binding protein 1:YB-1) phosphorylation in clinical treatment And its application method；

More specifically, the substance of directly or indirectly regulation YB-1 phosphorylation is also claimed in treatment inflammatory factor in the present invention Purposes in terms of associated diseases；It is further preferred that the present invention be also claimed YB-1 phosphorylation inhibitor treatment inflammatory because Purposes in terms of sub- associated diseases.

The invention includes using the individual of above-mentioned indication to be somebody's turn to do about the method in clinical application of YB-1 phosphorylation inhibitor Substance mitigates to treat, prevents above-mentioned indication.

The invention about the clinical application of the invention meaning substance include the invention meaning substance clinical use dosage form and Dosage.

Unless separately explaining, all scientific and technical terminologies have identical with essential term scientific and technological in field involved by the patent herein Meaning.Organic chemistry basic principle and specific functional groups and reaction reference Thomas Sorrell write " organic chemistry ", University Science Books, Sausalito:2006 version

Certain compounds of the invention perhaps exist in the form of specific geometry or stereochemical structure.Invention covering is all Such as such compound, including cis-, return formula, R and S correspond to isomers, non-corresponding isomers, (d) with (l) isomers, racemization Other mixtures that mixture and the invention are covered.It also include the replacement of asymmetric c atom, such as the replacement in alkyl.

Heterogeneous mixture includes the mixture of any ratio containing the invention isomers.

" the active ingredient effective quantity " of invention meaning refers to the dosage that can be enough to generate expected biological effect.This The effective dose that place refers to can according to different situations (such as administration route, individual difference, the medicine generation of different compounds, disease Type and therapeutic purpose) and it is different.

" treatment " of the disease of invention meaning includes curing, and mitigates, delays, or improves the method for the state of an illness, including to disease The prevention of feelings.Treatment may be one or more symptoms for disease, or lead to the pathology of symptom.Relative to same It is described herein to mitigate or prevent at least to show as 10% difference (with any standard technique Deng the control group for not receiving treatment It measures).

Signified " prevention " includes preventing herein, delays, avoids, or prevents the generation of disease or the state of an illness, is aggravated, or recurrence Method.

Signified " pharmaceutically acceptable auxiliary material " includes excipient herein, carrier, solvent, diluent, and in body The lapping of the medicine is transported in interior carrying.The example of these materials is such as: sugar, cellulose and its derivates, and west Huang alpine yarrow loses powder, Talcum powder, gelatin, oil, alcohols, agar, buffer, diethylester, emulsion, lubricant, no heat source water, alginic acid, toning and tune It is corresponding used in taste agent, preservative, emulsifier, humectant, lubricant, antioxidant, sustained release agent and other pharmaceutical formulations Substance.

It is of the present invention " separation or purifying " to refer to natural and storage the substance being substantially free of under normal conditions. Purity or homogeneity are determined by such as polyacrylamide cohesion electrophoresis or high performance liquid chroma- tography analytical chemistry methods.

Signified " individual " of the invention includes but are not limited to receive the mankind of the treatment, primate, rodent etc..It is " a Body " and " patient " can be interchanged for the mankind.

" dephosphorylation " i.e. mentioned by this paper " dephosphorylation ", refers generally to the removing of phosphate group, and herein contains It is adopted consistent with common meaning understood by one of ordinary skill in the art.

Unless otherwise defined, whole scientific and technical terminologies used herein have usually manages with those skilled in the art of the invention Solve identical meaning.For example, " autoimmunity disease " refers to autoimmune disease, refers specifically to body and autoantigen is occurred It is immunoreacted and leads to disease caused by damaged self tissue.Many diseases are listed in autoimmune disease in succession, are worth It proposes, the presence of autoantibody is not two concepts being equal with autoimmune disease, and autoantibody may be present in nothing The normal person of autoimmune disease especially the elderly, as anti-thyroglobulin antibody, Thyroid follicular epithelial cell antibody, Gastric parietal cell antibody, nucleus DNA antibody etc..Sometimes, it is damaged or the changed tissue of antigenicity can excite autoantibody It generates, when such as myocardial ischemia, downright bad cardiac muscle can lead to anti-myocardium autoantibody and be formed, but this antibody has no pathogenic effects, is A kind of secondary immune response.

The present invention is by taking the MCP-1 of one of YB1 target molecule as an example, animal experiment proves that YB1 phosphorylation is to inflammatory factor Regulating and controlling effect, the claimed drug of the present invention and the method for YB1 to treat inflammatory disease can be alleviated very significantly MCP 1 associated diseases illness, specifically, the drug of the invention can efficiently reduce the intracorporal disease of animal Interstitial lamella volume (area and thickness) at fat, inflammatory cell and atherosis in change arterial tissue, the drug can be efficient Inhibit or reduce the formation of atherosclerosis, and significantly suppresses the weight gain (table 3) of the experiment mice of High fat diet. The present invention confirms that drug of the invention may be up to absolutely efficacy for the efficiency assay result of animal. MCP-1 is one of the inflammatory factor of YB1 regulation.The present invention demonstrates the three kinds of different YB-1 that can most represent mechanism of the present invention Phosphorylation inhibitor (MK, YB-1 mutain (3ds), dexamethasone) acts on Ccl2 (i.e. MCP-1) mRNA downward consistent Property, and demonstrate they to lower Ccl2 (i.e. MCP-1), Ccl7, Serpine1, Ecm1, Serpinf1, Cd55, Il6, Uaca、Adora2b、 Tfrc、Cx3crl、Calcrl、Chi3l1、Mecom、Pde5a、Serping1、Il33、Ada、RT1- The function of the 27 class proinflammatory factor mRNA such as Db1, Ccl12, Ciita, Ccl11, Ace, Masp1, Tril, Acp5, Ghrl it is similar Property.According to these proinflammatory factors and the morbidity of related disease be associated with and the expression quantity of these proinflammatory factors declines to described Disease and its symptom bring are alleviated, mitigate these existing technologies facts, and the present invention proposes: using Targeted-control YB1 phosphorylation Substance come treat those YB1 as defined in the present invention regulation inflammatory factor participate in disease method or drug all wrap It includes within the protection that the present invention requests.

Detailed description of the invention

Fig. 1 human artery immunohistochemical staining, display YB-1 and phosphorylation YB-1 organize (AS) in human atherosclerotic Interior high expression, and the expression quantity in natural arterial tube wall (Normal) is extremely low.

The dyeing of the rat aorta oil red Europe Fig. 2.Compared with negative control group (DMSO), display MK2206 is significant (~85%) Inhibit the formation of atherosclerosis.Red area indicates the fat in atherosclerotic plaque.

Fig. 3 .C figure, arch of aorta frozen section oil red Europe dyeing, it is shown that MK significantly reduces the congee at the arch of aorta Sample hardenability.D figure, arch of aorta frozen section HE dyeing, it is shown that MK significantly reduces interstitial level at atherosis Product.

E-H figure, immunohistochemical staining respectively illustrate MK and significantly reduce MCP-1 (E) in atherosclerotic plaque, huge Phagocyte (F) and foam cells (G), and significantly reduce the YB-1 (H) of phosphorylation.(brownish red is positive staining area).

Fig. 4 .A figure: display V5 antibody (being purchased from Invitrogen/Life Technologies, Grand Island, NY) It can YB-1 mutain of the selective precipitation with V5 oligopeptides marker in immunoprecipitation experiment；(YBX is wild type YB-1 Same sense mutation；3dS is the deletion form of two adjacent amino acids of the 100th serine and its upstream), and wild type (WT) YB-1 is free of V5 oligopeptides marker, therefore cannot be precipitated by V5 antibody.

Note: IP=immunoprecipitation, IB (immunoblotting)=western blot hybridization, YB=YB-1 are used for Diagnosis of Sghistosomiasis The protein extract of mark hybridization is derived from the artery of wild type ductus arteriosus wall smooth muscle cell and stable transfection YBX and 3dS respectively Tube wall smooth muscle cell strain.

B figure: 4 width pictures are respectively to scheme i, ii, iii, iv from top to bottom；Wherein, figure i shows that MSP 14 (UK) can only (figure i, IB:UK western blot hybridization) is purified from protein extract by the V5 antibody in A figure together with YB-1 (YB). Moreover, the sediment containing UK and YB being purified out in immunoprecipitation has the activity of degradation MCP-1 mRNA.It reverses The biology of record-polymerase chain reaction (RT-PCR) immunoprecipitation obtained UK content and immunoprecipitate as the result is shown Activity (function of degradation MCP-1 mRNA) is directly proportional (figure ii).To on the compound middle addition UK antibodies block of the immunoprecipitation This bioactivity (figure iii) of degradation MCP-1 mRNA is stated, but compares PKC anti-δ then uninterrupted effect (figure iv).

Note: Extract=protein extract, rhUK=recombination human source UK114

Fig. 5 .A figure is the base variation schematic diagram of YB-1 muton.YB_mut=YB-1 muton.B figure is that reverse transcription-is poly- Polymerase chain (RT-PCR) reaction result: cell strain of the RNA from YB-1 muton stable transfection (fails to obtain S/A mutation The stable transfected cells strain of son) and wild type arterial smooth muscle cell.The cell of the wild and mutation containing YB-1 in culture exists It is incubated for 2 hours in culture solution containing 10 nanograms/milliliter PDGF (platelet derived growth factor), then as figure B is selectively added 1 micromole Dex (dexamethasone) is incubated for 2 hours.RNA derived from the above cell is used for RT-PCR.Foregoing YB- The specific primer of 1, MCP-1 and YB-1 muton is used for RT-PCR.Experimental result shows three kinds of YB-1 mutons It can be detected by RT-PCR with specific primer.There was only the MCP-1 of 3dS (serine deletion mutation) in three kinds of mutons MRNA content is minimum, and the MCP-1 mRNA of other two mutons is not significantly different with wild type.At dexamethasone The wild-type cell of reason contains only trace MCP-1 mRNA as 3dS.Phosphorylation in dexamethasone energy activating cell Enzyme, the latter make YB-1 dephosphorylation.Because dexamethasone is also to lower MCP-1 (see figure by lowering YB-1 phosphorylation 6), so being used as the positive control of this regulator control system in this experiment, (though dexamethasone can lower MCP-1, clinic is answered With showing that it has serious side effect and cannot be used for a long time).C figure be western blot hybridization (immunoblotting or Western Blot) result with detect wild-type rat same sense mutation smooth muscle cell (YBX) and containing YB1 mutation it is smooth Phosphorylation YB1 (pYB) is horizontal in muscle cell line (3dS).Phosphorylation YB1 has 50% or more in 3dS cell strain as the result is shown It lowers, i.e. the ratio that non-phosphorylating YB1 accounts for total YB1 (YB) dramatically increases.Above-mentioned these the results shows YB-1 is at it The important angle that the phosphorylation in 100 (be equal to people 102) sites is played the part of in the MCP-1 horizontal process in regulating cell Color.

Fig. 6 screens the signal transduction road that dexamethasone lowers MCP-1 effect.RT-PCR the result of A figure: rice is explicitly filled in Loose (Dex) significantly lowers MCP-1 mRNA (see the 2nd swimming lane), and the effect is (detailed to 6 kinds of kinases inhibitors used It is shown in Table 1) insensitive (see 3 to 8 swimming lanes).

RT-PCR the result of B figure: display dexamethasone (Dex) significantly lowers MCP-1 mRNA (see the 2nd swimming lane), and The effect can effectively be blocked (6 swimming lane) by a kind of phosphorglase inhibitor (Cal), but to other 5 kinds of phosphorylases used Inhibitor (see Table 1 for details) is insensitive (see 3,4,5,7,8 swimming lanes).The experiment indicates that dexamethasone is lowered by phosphorylase MCP-1.

Western blot hybridization (immunoblotting or Western Blot) result of C figure: YB-1 and phosphorylation are used The specific antibody of YB-1 finds that dexamethasone significantly lowers YB-1 (p-YB) water of phosphorylation by western blot hybridization Flat (the 2nd swimming lane).

Bioinformatic analysis result of Fig. 7 A. to RNA sequencing result.With the same sense mutation of wild type YB1 (YBX or YB1-V5 it is) control, the mRNA and known inflammatory factor lowered in YB1 muton (3dS) is selected from RNA sequencing result The inflammatory factor lowered to get 3dS of intersection.The comparison of the blue depth indicates the variation degree (>=3 of mRNA level in-site in figure Times).

Fig. 7 B.Quantitative reverse transcription polymerase chain reaction qRT-PCR lowers the substance of YB1 phosphorylation to one kind as the result is shown The similar inhibiting effect of inflammatory factor: there is 25 kinds in 27 kinds of mRNA that MK lowers 3dS quite similar downward to make With.Rat smooth muscle cell in culture is prepared after MK is stimulated for RNA, and resulting RNA is used for above-mentioned 27 kinds of mRNA's QRT-PCR detection.The comparison of the red depth indicates the variation degree (>=3 times) of mRNA level in-site in figure.

Specific embodiment

Further the contents of the present invention are described in detail below by specific embodiment, but this hair are not limited with this Bright protection scope.Unless otherwise specified, consumptive material used in following embodiments is commercially available；Operating procedure is normal Rule operation.

The source of biomaterial

Human body artery tissue used in the embodiment of the present invention and/or experimental example is derived from the clinical sample of BJ Union Hospital This；ApoE knocks out mouse (C57BL/6, ApoE-/-), 6 week old male C57BL/6, ApoE-/- mouse is purchased from Vital River, China (Beijing Vital River Experimental Animals Technology Co., Ltd.)；Ductus arteriosus wall smooth muscle cell strain is commercially available to be obtained ?.

Reagent and consumptive material

OCT compound (Tissue-Tek, IL1-9302) is purchased from Tissue-Tek company；

YB-1 antibody (Y0396) is purchased from sigma company；Secondary antibody (PV-6001) is purchased from company of Zhong Shan Golden Bridge；AEC(AEC- 0037) purchased from Newbiotics, Inc. advanced in years；Oil red O stain kit (ab150678) is purchased from U.S. abcam company (www.abcam.com)。

The total RNA extraction reagent box (RNeasy kit) that experimental example 3 uses is purchased from Qiagen Inc, Valencia, CA.

PKC anti-δ used in experimental example 4 is purchased from SigmaAldrich company；Anti- YB1 antibody (Y0396) is purchased from Sigma Aldrich；People UK recombinant protein (rhUK, H00010247-P01) and its antibody (00010247-M01) are purchased from Abnova (Littleton,CO)；V5 antibody is purchased from Invitrogen/Life Technologies, Grand Island, NY.

Dexamethasone used in experimental example 5 is commercially available.

RNA sequencing and its bioinformatic analysis are completed by Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.

The pharmaceutic usage of 1st group of embodiment, the present invention " the directly or indirectly substance of regulation YB-1 phosphorylation "

The substance that this group of embodiment provides directly or indirectly regulation YB-1 phosphorylation treats disease caused by inflammatory factor in preparation Application in terms of the drug of disease.

In the particular embodiment, the drug is using YB-1 as target spot；And the active constituent of the drug include directly or The substance of indirect adjustments and controls YB-1 phosphorylation, and reach disease caused by treatment inflammatory factor by the expression quantity of regulation inflammatory factor The purpose of disease.

In a more specific embodiment, the inflammatory factor is the inflammatory factor of YB-1 regulation；Caused by the inflammatory factor Disease includes: inflammation, diabetes, tumour, autoimmunity disease, immune defense, fat and encephalopathy；

In some embodiments, the active constituent of the drug includes directly or indirectly downward, and/or, directly or indirectly Inhibit the substance of YB-1 phosphorylation；

In further embodiments, the YB-1 phosphorylation inhibitor is selected from by any one of following substances or appoints several The group of composition: promote the substance of YB-1 dephosphorylation, the substance for blocking or delaying YB-1 phosphorylation, downward or inhibit YB-1's Substance, up-regulation or the substance for exciting YB-1 phosphatase activity of protein kinase activity.

The proinflammatory factor that the YB-1 phosphorylation inhibitor lowers expression be selected from Ccl2 (MCP1), Ccl7, Serpine1, Ecm1、Serpinf1、 Cd55、Il6、Uaca、Adora2b、Tfrc、Cx3crl、Calcrl、Chi3l1、Mecom、Pde5a、 Serping1、Il33、Ada、RT1-Db1、 Ccl12、Ciita、Ccl11、Ace、Masp1、Tril、Acp5、Ghrl。

In a further embodiment, the YB-1 phosphorylation inhibitor includes: substance shown in formula I:

Formulas I；And/or

Dexamethasone；And/or

YB-1 mutain；

In further specific embodiment, the active constituent of the drug further includes the YB-1 phosphorylation inhibitor Polymer, and/or, the YB-1 phosphorylation inhibitor pharmaceutically acceptable salt compounds；And/or esters chemical combination Object；And/or；Synergy class compound.

In a preferred embodiment, the drug further includes pharmaceutically acceptable auxiliary material and/or carrier.

2nd group of embodiment: drug of the invention

This group of embodiment provides a kind of drug for treating including inflammatory factor associated diseases.All implementation is organized at this In example, the drug all has following common trait: the drug is using YB-1 as drug target；And the drug activity at Dividing includes the substance that can directly or indirectly regulate and control YB-1 phosphorylation.

Drug provided by the present invention is as follows in the new anti-disease mechanism for the treatment of inflammatory factor associated diseases: intracellular non-phosphorus Acidification YB-1 can reduce intracellular nucleic ribosomal ribonucleic acid (mRNA) stability selectively to regulate and control monocyte chemoattractant protein-1 The expression of (monocyte chemotactic protein-1:MCP-1).YB-1 plays the mRNA stability of regulation MCP-1 Main function.(the dephosphorylation enhancing or phosphorylation for resulting from YB-1 subtract for the mildness increase of intracellular non-phosphorylating YB-1 It is weak, or both synergistic effect) expression of MCP-1 can be significantly inhibited, and then inhibit monocyte in local organization Gather, be finally reached inhibition, mitigate, or prevention is by inflammatory factor associated diseases.Thus, regulation YB-1 phosphorylation can be adjusted effectively It controls MCP-1 and then controls disease caused by be lacked of proper care by MCP-1.The atherosclerosis mould of present invention transgenic mice Type confirms the effect of the anti-disease mechanism.

In specific some embodiments, the meaning of " inflammatory factor associated diseases " mentioned by this paper are as follows: this field skill The pathogenesis documented in the prior art announced before the art personnel applying date according to the present invention (priority date) and morbidity The various diseases for thering is MCP 1 to participate in the process, specifically, comprising: inflammation (atherosclerosis, asthma Deng), diabetes B is fat, autoimmunity disease, tumour and encephalopathy (dull-witted, epilepsy etc.)；

And above-mentioned each disease type described herein " inflammation (atherosclerosis, asthma etc.), diabetes B are fat, Autoimmunity disease, tumour and encephalopathy (dull-witted, epilepsy etc.) ", the common skill of their own meaning and above-mentioned each disease areas The common meaning that art personnel are understood is consistent with range.

In other schemes of this group of embodiment, specifically, under the active constituent of the drug includes direct or indirect It adjusts, and/or, directly or indirectly inhibit the substance of YB-1 phosphorylation；

In a further embodiment, the direct or indirect downward, and/or, directly or indirectly inhibit YB-1 phosphorylation Substance include YB-1 phosphorylation inhibitor.The present invention original ground during the expression mechanisms of research regulation MCP-1 Have found effect and YB-1 phosphorylation of the YB-1 phosphorylation in regulation MCP-1 expression and the expression of other inflammatory factors The inhibiting effect that inhibitor expresses MCP-1.

In some embodiments, the YB-1 phosphorylation inhibitor is selected from by any one of following substances or appoints several form Group: can promote the substance of YB-1 dephosphorylation, the substance for blocking or delaying YB-1 phosphorylation, downward or inhibit YB-1 egg Substance, up-regulation or the substance for exciting YB-1 phosphatase of white kinases.Especially those highly selective directly result in YB-1 dephosphorization Acidification or the highly selective substance for directly blocking YB-1 phosphorylation and the highly selective protein kinase for lowering YB-1 (kinase) phosphatase (phosphatase) active substance of active substance and its highly selective up-regulation YB-1.

In above-described embodiment further embodiment, one of them specific example of the YB-1 phosphorylation inhibitor is such as Substance shown in Formulas I:

Formulas I.

Above-mentioned substance is that those skilled in the art's record according to the present invention and above-mentioned Formulas I can be obtained by artificial synthesized , alternatively, can be commercially available；Substance described in above-mentioned Formulas I is also referred herein as MK or MK2206.

Other than above-mentioned Formulas I, the YB-1 phosphorylation inhibitor can also be the drug of current clinical application: ground plug rice Pine；Dexamethasone can make YB-1 dephosphorylation by improving the activity of not yet determined phosphatase, to reduce thin MCP-1 in born of the same parents.The MCP-1 concentration of local organization be determine local organization in Monocytes/Macrophages quantity it is main because Element.Mouse atherosclerosis model shows that MCP-1 defect can make atherosclerosis decline 67% or so.

Another specific example of the YB-1 phosphorylation inhibitor is YB-1 mutain；The YB-1 mutain Refer to, by resulting mutain after the mutant serine in 102 site of amino acid sequence of wild type YB-1 albumen.Serine contains Hydroxyl is common phosphorylation site.The serine for removing or replacing the site can be such that YB-1 loses in the site by phosphoric acid The molecular basis of change, so the site can be no longer phosphorylated.The above-mentioned YB-1 mutain based on 102 site serines exists There is the function of lowering MCP-1 into the cell.In addition, the YB-1 polypeptide containing 102 serines loses the function of natural YB-1, but energy Protein kinase is competed with natural YB-1, and then reduces the probability that natural YB-1 is phosphorylated.Therefore, herein, this kind of mutation YB-1 be also attributed to YB-1 phosphorylation inhibitor, fall into the scope of YB-1 phosphorylation inhibitor of the present invention.

In some embodiments, the active constituent of the drug further includes the polymer of the YB-1 phosphorylation inhibitor, And/or the YB-1 phosphorylation inhibitor pharmaceutically acceptable salt compounds；And/or ester type compound；And/or； Synergy class compound.Those skilled in the art can according to actual needs, for example, drug system based on the content that the present invention records Make cost, pharmaceutical dosage form, raw material obtain convenience, drug except drug effect with for other properties (for example, stability, in vivo Disintegrating property etc.), select the derivative substance of Formulas I substance appropriate, that equivalent or similar drug effect can be reached.

In further embodiments, the drug further includes pharmaceutically acceptable auxiliary material and/or carrier.This field skill Art personnel can according to actual needs, for example, drug cost of manufacture, pharmaceutical dosage form, raw material obtain based on the content that the present invention records Take convenience, drug except drug effect with for other properties (for example, stability, disintegrating property in vivo, drug are guaranteed the quality Time limit etc.), auxiliary material appropriate is selected, specifically, including excipient, carrier, solvent, diluent, and to carry in vivo, Transport the lapping of the medicine.The example of these materials is such as: sugar, cellulose and its derivates, and west Huang alpine yarrow loses powder, talcum powder, Gelatin, oil, alcohols, agar, buffer, diethylester, emulsion, lubricant, no heat source water, alginic acid, toning and flavoring agent, prevent Respective substance used in rotten agent, emulsifier, humectant, lubricant, antioxidant, sustained release agent and other pharmaceutical formulations.

3rd group of embodiment: the screening technique of drug of the present invention

This group of embodiment provides a kind of screening technique of drug for treating inflammatory factor associated diseases.This organizes all realities Applying example all has following common trait: being carried out using the substance that can directly or indirectly regulate and control YB-1 phosphorylation as drug candidate Pathological experiment, and/or, clinical test, and/or, treatment is filtered out with effect, and/or generates the substance of curative effect as institute State the active constituent of drug.

In this group of some of which embodiment, the substance of the controllable YB-1 phosphorylation includes YB-1 inhibition of phosphorylation Agent；

This is organized in other embodiments, and the YB-1 phosphorylation inhibitor preferably is selected from: can promote the object of YB-1 dephosphorylation Matter, and/or, the substance of YB-1 phosphorylation is blocked or delays, and/or, the substance of the protein kinase of downward or inhibition YB-1, And/or raise or excite the substance of YB-1 phosphatase.

4th group of embodiment: the preparation method of drug of the present invention

This group of embodiment provides the substance that can lower YB-1 phosphorylation in preparation for treating inflammatory factor associated diseases Drug in terms of purposes；Or, for treating the preparation side of the drug of inflammatory factor associated diseases described in the 1st group of embodiment Method.This, which organizes all embodiments all, has following common trait: the substance for lowering YB-1 phosphorylation being placed on and is indicated In the commodity packaging case of inflammatory factor associated diseases therapeutical uses；

In this group of preferred embodiment, the combinable, inhibition, and/or the downward active substance of YB-1 include YB-1 Phosphorylation inhibitor；

In a still further embodiment, the YB-1 phosphorylation inhibitor is selected from: the substance of YB-1 dephosphorylation can be made, And/or the substance of YB-1 phosphorylation is blocked, and/or, lower the substance of the protein kinase of YB-1, and/or up-regulation YB-1 phosphorus The substance of sour enzyme.

5th group of embodiment: the application method of drug of the present invention

What drug described in this group of embodiment the 2nd group of embodiment of offer and/or the 3rd group of implementation case screening method screened The application method for the drug that the preparation method of drug and/or the 4th group of embodiment is prepared.This is organized all embodiments and all has Have the following characteristics that the substance that can directly or indirectly regulate and control YB-1 phosphorylation is preventing and/or treating inflammatory factor associated diseases side The purposes in face.

In some embodiments, indication and its user of the specific inhibitor that YB1 phosphorylation is provided in clinical treatment Method；

In other embodiments, the application method includes the clinical application of the drug, that is, including to above-mentioned adaptation The individual of disease, to treat, is mitigated using the substance, prevents above-mentioned indication.

In a further embodiment, the clinical application of the drug includes the clinical use dosage form of the invention meaning substance And dosage.

Experimental example 1, the test of human artery's immunohistochemical staining

The fresh arterial tissue of people is embedded in OCT compound (Tissue-Tek, IL1-9302), the ice after liquid nitrogen quickly cooling Freeze slice and is stored in -80 degree.6 microns of slabs are carried out with the immunohistochemical staining of standard.The immunohistochemical staining side " the Immunohistochemistry Protocol that the operation of method is announced with reference to " cellsignal company " official website (Frozen) " operating method (network address: https: //www.cst-c.com.cn/contents/resources- protocols/immunohistochemistry-protocol-(frozen)/ihc-frozen).In addition, this experiment uses Sheep blood serum (Zhong Shan Golden Bridge, ZLI-9056) closes non-specific background.Primary antibody is respectively anti-YB-1 antibody (sigma, Y0396) With anti-phosphorylation YB-1 antibody (Cell Signaling, C34A2), incubated afterwards with biopsy tissues at 4 degree with PBS dilution (1:100) It educates overnight.Secondary antibody is purchased from Zhong Shan Golden Bridge (PV-6001).For AEC (stepping novel agent AEC-0037) for developing the color, red is the positive Area.

Experimental result shows that YB-1 and phosphorylation YB-1 organizes the height in (AS) to express in human atherosclerotic, and Expression quantity in natural arterial tube wall (Normal) is extremely low.

Experimental example 2, the test of rat aorta oil red O stain

To Atherosclerosis Model mouse commonly used in the art using any drug of the 1st group of embodiment of the invention It is treated, administration mode: 85 micro- grams/day of intraperitoneal injection；Control group mice uses the DMSO of equivalent daily；After administration 70 days, The arterial tissue of the mouse of mouse and control group to treatment group is respectively adopted oil red O stain kit and dyes, dyeing Method is that this field shows most common standard method fatty in frozen section tissue, and concrete operation step is according to purchased from beauty The product description of the oil red O stain kit (ab150678) of abcam company of state (www.abcam.com).

Test result is shown: as shown in Fig. 2, display MK significantly suppresses Atherosclerosis compared with control group (DMSO) The formation of change.Red area indicates the fat in atherosclerotic plaque.

" Atherosclerosis Model mouse " herein, " model mice " refer to the C57BL/6 after High fat diet 10 weeks, N9, ApoE^-/-Mouse.

The effect of experimental example 3, medical treatment atherosclerosis of the present invention

First using western blot hybridization (IB) and reverse transcription-polymerase chain reaction (RT-PCR) detection MK in molecular water Influence of the gentle protein level to MCP-1；Concrete operations are；Mouse and human smooth muscle cell (mSMC, hSMC) are containing It is incubated for 4 hours in 0.5 micromolar MK2206 culture solution, the cell being incubated in no MK2206 culture solution is as control (C). Albumen and ribonucleic acid are extracted from above-mentioned cell, and YB-1 and MCP-1mRNA therein are detected for IB and RT-PCR and is contained Amount；The operating method of IB and RT-PCR is carried out by routine operation well known to those skilled in the art.Wherein reverse transcription-polymerase The concrete operations of chain reaction (RT-PCR) are as follows:

The primer of use is respectively the 340-625 core of the 145-595 nucleic acid encode section and GAPDH of crossing over rat MCP-1 Acid encoding section；Primer length is that (the nucleic acid encode region sequence of rat MCP-1 and GAPDH can log in 20 bases Genebank inquiry is known).PCR product is the 145-595 of rat monocyte chemoattractant protein cDNA sequence respectively；GAPDH's 340-625；The 420-988 of YB-1；The 441-988 of YB-1 muton.Wherein, the forward primer of YB-1 muton is TGTGGAATTCGACGTCGTC corresponds to wild type sequence TGTGAGTTTGATGTTGTT.Unless separately explaining, above-mentioned primer is suitable All RT-PCR for this paper.

Total serum IgE (Total RNA) is extracted with RNeasy kit (Qiagen Inc, Valencia, CA) from rat cell. Rat smooth muscle cell isolation and culture is shown in Brock, 1985, Hypertension, it operates substantially as follows: 200-300 grams of male Smooth muscle cell in the artery in the heart of Sprague-Dawley rat is separated through enzymatic isolation method to be obtained.All experiments use 5-14 The culture cell in generation.Cell culture is in the Dulbecco modified Eagle medium (DMEM for containing 10% calf serum； Gibco Laboratories, Gaithersburg, Md.) in culture solution, it is incubated in 37 degree of carbon dioxide incubators.Work as culture dish In cell reach about 60% it is full when, cell be used to prepare RNA.Cell transfecting presses Lipofectamine2000 reagent system The operating method of quotient Ivitrogen is made, Lipofectamine2000 is purchased from Invitrogen.Cell density when transfection is about 30%.Each reverse transcriptase chain reaction uses 200 nanogram RNA.Reaction system uses Masterscript RT- PCR system(5PRIME Inc., Gaithersbueg,MD).RT-PCR response procedures are as follows:30 minutes；2 points Clock；With "22 seconds,22 seconds,44 seconds " it is 1 circulation, 27 circulations are carried out altogether；6 minutes.Unless separately doing Illustrate, the above parameter is suitable for all RT-PCR reactions herein.

Unless otherwise specified, " YB-1 muton " herein refers generally to: " the YB-1 mutain " corresponding core Nucleotide sequence.

Testing result is as shown in the A figure of Fig. 3: western blot hybridization (IB) and reverse transcription-polymerase chain reaction (RT- PCR), display MK2206 (the protein kinase specific inhibitor of MK, YB-1) can efficiently inhibit vascular smooth muscle and monocyte Interior YB-1 phosphorylation and MCP-1mRNA are horizontal.

Atherosclerosis Model mouse is treated using any drug of the 1st group of embodiment of the invention, is given Prescription formula: while to experimental mouse high fat diet, 85 micro- grams/day of intraperitoneal injection (treats tumor dose about in mouse model 1/13.See what Hirai et al. was deliveredMol Cancer Ther.2010 Jul；9(7):1956-67.)；Control group mice is daily Drug of the invention is replaced using the DMSO of equivalent；After being administered 10 weeks, the artery of the mouse of mouse and control group to treatment group Oil red O stain and artery HE dyeing is respectively adopted in tissue, and the concrete operations of the oil red O stain are as described in experimental example 2；Artery HE dyeing refers to HE (hematoxylin-eosin) colouring method, and concrete operations are announced referring to the official website " protocolsonline " " Haematoxylin Eosin (H&E) staining | Protocols Online " operating method (official's network address:

Http:// www.protocol-online.org/cgi-bin/prot/view_cache.cgi? ID=2503). HE dyeing be the most common Standard histological colouring method in this field in the form of showing tissue and structure (hematoxylin be it is dark blue Or purple, in conjunction with nucleic acid.Yihong is in pink colour, in conjunction with amino acid)；The B-D of therapeutic effect such as Fig. 3 of this group test schemes institute Show: model hero mouse aorta oil red O (Oil Red O) dyeing, display MK2206 efficiently inhibit model hero mouse Atherosclerosis The formation of change.(DMSO is no medicine group, 6 week old male C57BL/6, ApoE^-/-Mouse was through High fat diet 10 weeks；MK is administration group: 6 week old hero C57BL/6, ApoE^-/-Mouse is administered simultaneously 10 weeks through High fat diet, occurs red region in figure for display arterial blood Fat in tube wall atherosclerotic lesion).C figure, arch of aorta oil red Europe dyeing, it is shown that MK is significantly reduced in aorta Atherosis degree at bow.D figure, artery HE dyeing, it is shown that MK significantly reduces interstitial lamella area at atherosis.

Meanwhile this experimental example also is reduced by inflammation, reduces inflammatory cell for drug of the invention in terms of test, control group and The therapeutic administratp process of administration group animal is as described by the preceding paragraph, to the artery of control group and administration group mouse after treatment end Tissue carries out immunohistochemical staining respectively, and method is the same as experimental example 1；The E-H of coloration result such as Fig. 3 schemes, immunohistochemical staining, point Do not show that MK is significantly reduced the MCP-1 in atherosclerotic plaque (E), macrophage (F) and foam cells (G), and Significantly reduce the YB-1 (H) of phosphorylation.(brownish red is positive staining area).

Atherosclerosis is mainly the inflammation by the MCP-1 arterial blood tube wall mediated.Above-mentioned experimental example is athero- with artery It is hardened to model, by taking MCP-1 as an example, demonstrates effect of YB-1 during inflammatory pathologies, and by with of the invention Drug regulation YB-1 phosphorylation regulates and controls antiphlogistic effects caused by internal MCP-1 level in turn.In addition to MCP-1, IL6 etc. The proinflammatory factor of YB1 regulation is also the factor for inspiring atherosclerosis.The Anti-inflammatory Mechanism of the YB1 illustrated according to the present invention With the mechanism of action of drug of the present invention, drug of the present invention equally can be used for treating by the inflammation of MCP-1 and other YB1 regulation The diabetes B of the mediated generation of sex factor, tumour, autoimmunity disease, immune defense, fat and encephalopathy (dull-witted, epilepsy Deng) etc. illnesss, and expectability obtains the therapeutic effect similar with this paper experimental example 2 and 3.

Experimental example 4, YB-1 mutain inhibit the verifying of MCP-1 activity

First by immunoprecipitation assay, the YB-1 mutain with V5 oligopeptides marker is selected using V5 antibody Selecting property precipitating.Concrete operations are as follows:

YB-1 mutain obtains in the following way: using RT-PCR as described in experimental example 3 concrete operations (including Using YB-1 muton primer, response procedures etc.), to make rat YB-1 (GEnBank accession no.NM031563) CDNA across starting and termination codon area is obtained through reverse transcriptase chain reaction, and is cloned in pBluescript Between the HindIII and XbaI of II KS (+) plasmid.All YB-1 mutains all use polymerase chain reaction (PCR) raw At or other artificial sequences synthesize to obtain.These YB-1 mutons are subcloned on pcDNA3.1/V5-His A plasmid Initiation codon upstream all introduces Kozak sequence, GCCACC between the site Hind III and XbaI.The end YB-1 3 ' Termination codon replaced by Xba I site.The label peptide of V5-His contained in plasmid and termination codon are followed closely by coded sequence After Xba I site.The PCR final product of clone is all confirmed through DNA sequencing.In the Rat Smooth Muscle for expressing the YB1 mutain Into the cell, the content of phosphorylation YB1 is than in wild-type rats smooth muscle cell

YB-1 mutain with V5 oligopeptides marker obtains in the following way: pcDNA3.1/V5-His A carrier DNA sequence dna of the plasmid with V5 label peptide, is cloned into V5's for frame (in frame) is entered without the YB-1 mutant of termination codon Hybrid plasmid is just obtained after upstream.This hybrid plasmid can express the YB-1 mutain with V5 in the cell.It is this miscellaneous The mutain of conjunction can be in conjunction with V5 antibody specificity.

The specific primer and RT-PCR reaction interval of YB-1, MCP-1 and YB-1 muton as described in experimental example 3 Sequence be used to obtain in the RT-PCR test of RT-PCR result shown in the B figure of corresponding diagram 5.

Unless separately explain, immunoprecipitation herein, the operating procedure of western blot hybridization are as follows: each sample Use 30 microlitres of G-protein pearl suspensions (Roche, Germany).With the S100 buffer of pre-cooling (10mM Tris [pH 7.4], 1.5mM MgCl2,150mM KCl, 0.5 mM DTT, 0.5mM PMSF) it washes G-protein pearl 3 times.Again with 3 microlitres of V5- antibody and G Albumen pearl is incubated for 1 hour in 4 C, then adds 1 microlitre of 20% bovine serum albumin(BSA) (BSA Fraction V from Sigma Aldrich) continue to be incubated for 1 hour.It is primary that the G-protein pearl that V5 antibody has been coated with is washed with the S100 buffer of pre-cooling, is then used 150 microlitres of cold S100 buffers are resuspended in ice bath in 0.5 milliliter of Eppendorf pipe.30-50 microlitres (10 micrograms of protein) is added (method for extracting refers to Poon M, Liu B, Taubman MB.1999. Identification of a to endochylema extract novel dexamethasone-sensitive RNA-destabilizing region on rat monocyte 1 mRNA.Molecular and cellular biology 19:6471-6478 of chemoattractant protein) in(sample containing recombinant human UK albumen is needed 50 microlitres of endochylema extracts in 1 microlitre of recombined human within 1 hour for incubation altogether UK albumen is incubated in incubation at room temperature 5 minutes, then with the coated G albumen pearl of V5 antibody).Immune precipitation is centrifuged with 110x g Object 1 minute to collect G-protein pearl, with S100 buffer wash gained G-protein pearl 2 times, be resuspended in 30 microlitres of S100 buffers in case For RNA degradation experiment, (5 microlitres=1 microgram RNA and 5 microlitres of G-protein pearl re-suspension liquids are incubated for 5 minutes altogether in room temperature, take 2 microlitres Supernatant is in 20 microlitres of reverse transcriptase chain reaction) or 30 microlitres of sample-loading buffer (22 l RIPA 8 l of buffer plus, 4 loading dye:160mM Tris pH 6.8,4%SDS, 40%Glycerol, 575mM β- Mecapital ethanol, 20 μ l 2%Xylene Cyanol), it is subsequently used in western blot hybridization experiment:5 minutes, Eluent is used for 4-20%SDS-PAGE (polyacrylamide gel electrophoresis), by the protein delivery in gel after electrophoresis completion Onto pvdf membrane, then with detection of specific antibody destination protein.

Test result difference is as shown in Figure 4 and Figure 5.Control PKC anti-δ used in figure iv in the B figure of Fig. 4 is a kind of Protein kinase, the antibody specificity combination people, mouse and rat this protein kinase, the antibody to MCP-1 mRNA degrade The uninterrupted effect of activity；The serine in 102 sites of YB -1 is equivalent to the serine in 100 site of rat.In rat YB-1 In, the missing of the serine causes YB-1 that cannot be phosphorylated in the site.YB-1 and UK114 of the site without phosphorylation (a kind of ribalgilase) and GR (glucocorticoid receptor) are combined into activated complex, the activated complex degradation selectivity MCP-1 mRNA.It is unfavorable for combining UK114 in the YB-1 of the site phosphorylation.

MCP-1 mRNA content in three kinds of mutons in Fig. 5 in only 3dS (serine deletion mutation) cell is most It is low, and the MCP-1 mRNA of other two mutons is not significantly different with wild type, meanwhile, the phosphorylation in 3dS cell YB1 level is decreased obviously, that is to say, that non-phosphorylating YB1 level is significantly increased under the premise of overall YB1 is poor (Fig. 5 C).The results show YB -1 its 100 (being equal to people 102) site phosphorylation in regulating cell The key player played the part of in MCP-1 horizontal process.

Experimental example 5, dexamethasone inhibit MCP-1 activity and lower the verifying of YB-1 phosphorylation

It is also to be lowered by lowering YB-1 phosphorylation that this experimental example, which equally uses RT-PCR verification experimental verification dexamethasone, MCP-1.

Primer used in RT-PCR test and specific experiment operating procedure are as described in experimental example 3.And used in testing The information of other 6 kinds of phosphorglase inhibitors is detailed in the following table 1:

Table 1. be used for screen mediate dexamethasone lower MCP-1 effect signal transduction road used in protein kinase with The use concentration of phosphorglase inhibitor and these reagents, target molecule and inhibitory effect

Note: the concentration in upper table refers to the final concentration of each inhibitor in cell culture medium；+: complete inhibition；+/-: part presses down System；: do not inhibit

Test result is as shown in Figure 6, the results showed that: dexamethasone (Dex) significantly lowers MCP-1 mRNA, and prompts ground This effect of Sai meter Song is realized by activation phosphatase.

The zoopery validity statistics of experimental example 6, drug of the present invention

Using 24 model mices, control group and administration group each 12, according to the administration mode as shown in experimental example 3 and right Administration group and control group are administered respectively according to mode or equivalent DMSO, administration refer to 50 mouse peritoneals note to administration group Penetrate drug described in the 1st group of embodiment any embodiment of the invention；After 10 weeks, using the oil red O (Oil as shown in experimental example 3 Red O) it dyes and the arterial tissue of two groups of mouse is dyed respectively with immunohistochemical staining, finally obtain experimental example 2-3's Treatment results (to save length, no longer enumerate relevant coloration result figure and statistical graph) one by one herein, and every of administration group The formation of the atherosclerosis of mouse is significantly inhibited, atherosis degree is substantially reduced, interstitial lamella body at atherosis (area and thickness) is substantially reduced product, inflammatory cell substantially reduces, inflammation is significantly alleviated, and therefore, drug of the invention exists Validity in animal experiment is 100%.

Experimental example 7, YB-1 phosphorylation inhibitor are to the inhibiting effect of 27 kinds of proinflammatory factors

This experimental example is sequenced using RNA and quantitative reverse transcription polymerase chain reaction qRT-PCR, demonstrates YB-1 phosphorylation Downward of the inhibitor (MK, YB1 muton (3dS)) to 27 kinds of proinflammatory factors, inhibitory effect.RNA sequencing and quantitative reverse transcription are poly- Synthase chain reaction qRT-PCR can be used Conventional procedures well-known to those skilled in the art and carry out.

The result shows that: MK (inhibiting YB1 phosphorylation) and dexamethasone (promoting YB1 dephosphorylation) are in vascular smooth muscle cells The ultimate effect of YB1 is all the YB1 for generating non-phosphorylating, that is, the YB1 of intracellular non-phosphorylating is caused to increase.In this regard with The case where YB1, is the same in 3dS cell strain and non-phosphorylating YB1 increases.So the present invention only to the transcript profile of 3dS cell into The RNA that gone is sequenced and analyzes the mRNA variation of wherein inflammatory factor, and original discovery non-phosphorylating YB1 lowers 27 kinds of inflammation The level of the mRNA of sex factor in the cell.Intracellular mRNA level in-site depends on the mRNA stability after transcribing and turning green.YB1 Influence to MCP1mRNA is to lower its stability, but be not excluded for the possibility for having an impact transcription to other inflammatory factors.3dS is only Increase the content of intracellular non-phosphorylating YB1；And the content of MK and dexamethasone in addition to increasing intracellular non-phosphorylating YB1, also Other albumen except YB1 are targeted, these are also possible to by other albumen that MK and ground plug rice regulate and control through machine after transcription or transcription System is to adjust the mRNA level in-site of other inflammatory factors, or has Different Effects to above-mentioned inflammatory factor.According to 3dS, MK and ground plug Meter Song equally lowers the fact that MCP1 mRNA stability, can estimate MK and dexamethasone lower YB1 27 in logic The mRNA of kind inflammatory factor has roughly the same effect.27 kinds of inflammatory factors that MK lowers 3dS are verified by qRT-PCR Effect, it was confirmed that MK have to the effects of these inflammatory factors and the effect of 3dS 93% similitude (in addition to Serpinf1 and Tfrc is not lowered other than effect, has similar downward to act on remaining 25 kinds of inflammatory factor.See Fig. 7 B).Dexamethasone is The known drug for lowering MCP1, the positive control substance for lowering MCP1 is made in the embodiment of the present invention, concurrently of dexamethasone Its existing effect is by lowering YB1 phosphorylation.Dexamethasone acts on inhibition, the downward of the YB1 27 class proinflammatory factors lowered Similar effect as shown in figs. 7 a-b can be obtained, repeats no more herein.

The inflammatory factor of table 2.YB1 phosphorylation and the relationship of disease

In upper table 2 ,+: have commercialized clinical or experimental drug object；Most right column is corresponding reference numbers.This In a little bibliography, discloses relevant experimental data and confirm that related disease can be improved by lowering these above-mentioned inflammatory factors Symptom.Inhibit used in document the expression of these inflammatory factors method include: gene knockout or mutation, specific antibody, And antagonist etc., but be not directed to using YB-1 phosphorylation inhibitor.

The Bariatric effect of experimental example 8, YB-1 phosphorylation inhibitor

This experimental example demonstrates YB-1 phosphorylation inhibitor to fat inhibition (treatment) effect, with original body mass 20-22 Gram experimental mouse (ApoE-/-) be experimental subjects, High fat diet, administration group (Drug) is using the processing of YB-1 phosphorylation inhibitor Experimental mouse, and control group (DMSO) then handles experimental mouse, processing mode and process cycle and 6 phase of experimental example with the DMSO of equivalent Together.

	Weight g
		Drug
l#	20.94
		2#	26.5
3#	24.9
		4#	27.1
5#	25.7
		Average value	25.028
sd	2.431032702
		p	0.088658742


DMSO
		6#	27.52
7#	26.8
		8#	27.71
9#	28.4
		10#	26.7
11#	27.6
		Average value	27.455
sd	0.629658638

The weight data of table 3. administration group and control group mice measured at the end of testing.

Table 3 shows that MK significantly suppresses the increase (20-22 grams of original body mass) of experimental mouse weight, and P value > 0.05 has statistics Learn meaning.Table 3 show MK to fat or weight gain inhibiting effect, obesity be also inflammatory factor such as Serpine1, Disease caused by the contour expression of Serpinf1.MK significantly lowers the mRNA level in-site of Serpine1, therefore inhibits obesity.Table 3 Middle control group has 10% weight gain, and this statistically significant weight gain shows MK to fat or weight inhibition Effect.

The YB-1 muton (3ds) that above-mentioned experimental mouse intraperitoneal injection (once a week) slow virus (Lentivims) is carried The similar remarkable result of table 3 as above can be also obtained in terms of inhibiting mouse weight to increase (obesity).

SEQUENCE LISTING

<110>Jilin Zhong Tai Bioisystech Co., Ltd

<120>directly or indirectly regulate and control the substance of YB-1 phosphorylation in terms of the drug of preparation treatment inflammatory factor associated diseases Application

<130> P180462/JLZ

<150> 201710657706.9

<151> 2017-08-03

<150> 201810175137.9

<151> 2018-03-02

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 324

<212> PRT

<213> Artificial Sequence

<220>

<223>wild type YB-1 protein sequence

<400> 1

Met Ser Ser Glu Ala Glu Thr Gln Gln Pro Pro Ala Ala Pro Pro Ala

1 5 10 15

Ala Pro Ala Leu Ser Ala Ala Asp Thr Lys Pro Gly Thr Thr Gly Ser

20 25 30

Gly Ala Gly Ser Gly Gly Pro Gly Gly Leu Thr Ser Ala Ala Pro Ala

35 40 45

Gly Gly Asp Lys Lys Val Ile Ala Thr Lys Val Leu Gly Thr Val Lys

50 55 60

Trp Phe Asn Val Arg Asn Gly Tyr Gly Phe Ile Asn Arg Asn Asp Thr

65 70 75 80

Lys Glu Asp Val Phe Val His Gln Thr Ala Ile Lys Lys Asn Asn Pro

85 90 95

Arg Lys Tyr Leu Arg Ser Val Gly Asp Gly Glu Thr Val Glu Phe Asp

100 105 110

Val Val Glu Gly Glu Lys Gly Ala Glu Ala Ala Asn Val Thr Gly Pro

115 120 125

Gly Gly Val Pro Val Gln Gly Ser Lys Tyr Ala Ala Asp Arg Asn His

130 135 140

Tyr Arg Arg Tyr Pro Arg Arg Arg Gly Pro Pro Arg Asn Tyr Gln Gln

145 150 155 160

Asn Tyr Gln Asn Ser Glu Ser Gly Glu Lys Asn Glu Gly Ser Glu Ser

165 170 175

Ala Pro Glu Gly Gln Ala Gln Gln Arg Arg Pro Tyr Arg Arg Arg Arg

180 185 190

Phe Pro Pro Tyr Tyr Met Arg Arg Pro Tyr Gly Arg Arg Pro Gln Tyr

195 200 205

Ser Asn Pro Pro Val Gln Gly Glu Val Met Glu Gly Ala Asp Asn Gln

210 215 220

Gly Ala Gly Glu Gln Gly Arg Pro Val Arg Gln Asn Met Tyr Arg Gly

225 230 235 240

Tyr Arg Pro Arg Phe Arg Arg Gly Pro Pro Arg Gln Arg Gln Pro Arg

245 250 255

Glu Asp Gly Asn Glu Glu Asp Lys Glu Asn Gln Gly Asp Glu Thr Gln

260 265 270

Gly Gln Gln Pro Pro Gln Arg Arg Tyr Arg Arg Asn Phe Asn Tyr Arg

275 280 285

Arg Arg Arg Pro Glu Asn Pro Lys Pro Gln Asp Gly Lys Glu Thr Lys

290 295 300

Ala Ala Asp Pro Pro Ala Glu Asn Ser Ser Ala Pro Glu Ala Glu Gln

305 310 315 320

Gly Gly Ala Glu

Claims

1. the directly or indirectly substance of regulation YB-1 phosphorylation answering in terms of the drug of preparation treatment inflammatory factor associated diseases With.

2. application according to claim 1, which is characterized in that the drug is using YB-1 as target spot；And the work of the drug Property ingredient include direct or indirect regulation YB-1 phosphorylation substance, and the expression quantity by regulating and controlling inflammatory factor reaches treatment The purpose of inflammatory factor associated diseases.

3. application according to claim 1 or 2, which is characterized in that the inflammatory factor is the inflammatory factor of YB-1 regulation； The inflammatory factor associated diseases include: inflammation, diabetes, tumour, autoimmunity disease, immune defense, fat and encephalopathy；

The inflammation is preferably atherosclerosis, asthma, rheumatic arthritis；The encephalopathy preferably is selected from dementia, epilepsy, depression Disease.

4. application according to claim 1 to 3, which is characterized in that the active constituent of the drug include directly or Downward is connect, and/or, directly or indirectly inhibit the substance of YB-1 phosphorylation；

Further, the direct or indirect downward, and/or, directly or indirectly inhibiting the substance of YB-1 phosphorylation includes YB-1 Phosphorylation inhibitor；

5. application according to claim 4, which is characterized in that the YB-1 phosphorylation inhibitor is in by following substances It is any or appoint several compositions group: promote the substance of YB-1 dephosphorylation, the substance for blocking or delaying YB-1 phosphorylation, under Substance, up-regulation or the substance for exciting YB-1 phosphatase activity of the protein kinase activity of tune or inhibition YB-1；

The YB-1 phosphorylation reinforcing agent is selected from by any one of following substances or appoints several groups formed: blocking or delays The substance of YB-1 dephosphorylation, up-regulation excite the substance of YB-1 phosphorylation, up-regulation or the protein kinase activity for exciting YB-1 Substance, inhibition or the substance for lowering YB-1 phosphatase activity；

The proinflammatory factor that the YB-1 phosphorylation inhibitor lowers expression be selected from MCP-1, Ccl2, Ccl7, Serpine1, Ecm1, Serpinf1、Cd55、Il6、Uaca、Adora2b、Tfrc、Cx3crl、Calcrl、Chi3l1、Mecom、Pde5a、 Serping1、Il33、Ada、RT1-Db1、Ccl12、Ciita、Ccl11、Ace、Masp1、Tril、Acp5、Ghrl。

6. application according to claim 4 or 5, which is characterized in that the YB-1 phosphorylation inhibitor includes: such as Formulas I institute The substance shown:

Dexamethasone；And/or

YB-1 mutain；

The YB-1 mutain refers to, obtains after the 102 site serines mutation of the amino acid sequence of wild type YB-1 albumen Mutain.

7. according to any application of claim 4-6, which is characterized in that the active constituent of the drug further includes described The polymer of YB-1 phosphorylation inhibitor, and/or, the YB-1 phosphorylation inhibitor pharmaceutically acceptable salt chemical combination Object；And/or ester type compound；And/or；Synergy class compound.

8. -7 any application according to claim 1, which is characterized in that the drug further includes pharmaceutically acceptable auxiliary Material and/or carrier.

9. a kind of screening technique for the drug for treating inflammatory factor associated diseases, which is characterized in that using directly or indirectly to adjust The substance for controlling YB-1 phosphorylation carries out Pathological experiment as drug candidate, and/or, clinical test, and/or, treatment, screening is provided It is effective, and/or generate active constituent of the substance as the drug of curative effect.

10. screening technique according to claim 8, which is characterized in that described directly or indirectly to regulate and control YB-1 phosphorylation Substance include YB-1 phosphorylation inhibitor；

The YB-1 phosphorylation inhibitor preferably is selected from: the substance of YB-1 dephosphorylation can be promoted, and/or, block or delay YB-1 The substance of phosphorylation, and/or, the substance of the protein kinase activity of downward or inhibition YB-1, and/or, up-regulation or excitation YB-1 The substance of phosphatase activity.