CN101919844A - Compound for controlling autoimmune disease and use thereof - Google Patents

Abstract

The invention relates to a compound for controlling autoimmune disease and use thereof, wherein, the compound comprises berberine and pharmaceutically acceptable salt or a derivative thereof, and the autoimmune disease refers to type I diabetes or insulitis and the like. The invention further provides the new use of the berberine for the first time; and the berberine has strong pharmacological effects, no toxic side effects and good pharmaceutical prospect.

Description

Chemical compound of control autoimmune disease and uses thereof

Technical field

The invention belongs to pharmaceutical field, more specifically, the present invention relates to chemical compound of preventing and treating autoimmune disease and uses thereof.

Background technology

Diabetes are divided type i diabetes (Type I Diabetes) and type ii diabetes (Type II Diabetes) and gestational diabetes (Gestational Diabetes) usually.Type i diabetes is different fully with the pathogenesis of type ii diabetes.Wherein the type i diabetes pilosity is born in teenager, and its insulin secretion lacks, and must rely on insulinize and earn a bare living.During type ii diabetes is more common in after 30 years old, the old people, its secretion of insulin amount is not low even also higher, the cause of disease mainly is that body is to insulin insensitivity (being insulin resistant).

Think that at present type i diabetes is a kind of autoimmune disease, under multiple factor combined effect, body to insulin multiple antigenic component immunity owe tolerance, finally caused the absolute shortage of insulin.The main treatment means of type i diabetes is that lifelong insulin injection or insulin analog come glucose level control at present.Treatment means to type i diabetes had also obtained certain breakthrough in recent years, provided new thinking and method as the research of the aspects such as decoding of xenotransplantation, novel insulin and Drug therapy, type i diabetes vaccine, genetic immunization treatment and Disease-causing gene for type i diabetes.

In case suffer from " diabetes ", will reduce 10 years more than life-span, and contingent complication spread all over whole body.In recent years, Chinese diabetes prevalence significantly increases.At present the diabetics number surpasses 6,000 ten thousand, accounts for 1/5th of world's diabetic population sum, and prevalence occupies the second place of the world, and with every days at least 3000 people speed increase.

Therefore, develop effective medicine and become the problem that this area presses for solution with control of diabetes.

Summary of the invention

The object of the present invention is to provide chemical compound of control autoimmune disease and uses thereof.

In a first aspect of the present invention, the purposes of berberine, its pharmaceutically acceptable salt or derivatives thereof is provided, be used to prepare prevention, treat or improve the compositions of autoimmune disease.

In a preference, the pharmaceutically acceptable salt of described berberine is the hydrochlorate of berberine.

In another preference, described autoimmune disease is Th1 or the relevant autoimmune disease of Th17 cell.

In another preference, described autoimmune disease is a type i diabetes.

In another preference, described compositions also is used for: prevention or treatment insulitis.

In another preference, described compositions also is used for: the generation of the inflammation-inhibiting factor; Or the generation of promotion anti-inflammatory factors.

In another preference, described inflammatory factor is selected from: IL-17, IL-6, IFN γ or TNF α.

In another preference, described anti-inflammatory factors is selected from: IL-4 or IL-10.

In another preference, described compositions also is used for: the Th17 differentiation of suppressor T cell, promptly suppressor T cell is to the Th17 cell differentiation.

In another preference, described compositions also is used to activate Erk.

In another preference, described compositions is by activating the Th17 differentiation of Erk suppressor T cell.

In another preference, described compositions also is used for: the Th1 differentiation of suppressor T cell, promptly suppressor T cell is to the Th1 cell differentiation.

In another preference, described compositions also is used to reduce the activity of p38 or JNK.

In another preference, described compositions is come the Th17 differentiation of suppressor T cell by the activity that suppresses p38 or JNK.

In another preference, described compositions also is used for: the protein expression level that reduces STAT4; Or increase the ubiquitinization of STAT4.

In another preference, described compositions reduces the protein expression level of STAT4 by Ubiquitin-Proteasome Pathway.

On the other hand, a kind of prevention, treatment are provided or improve the method for autoimmune disease, described method comprises, needs to prevent, treat or improve berberine, its pharmaceutically acceptable salt or derivatives thereof of the object effective dose of autoimmune disease.

In another preference, described to liking mammal.More particularly, described mammal is the people.

Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.

Description of drawings

Fig. 1, berberine have reduced the sickness rate of diabetic mice.

The female NOD mouse stomach in 11 weeks was to berberine hydrochloride totally 14 days.A: measure random blood sugar weekly, can be regarded as diabetes when blood glucose is higher than 250mg/dL.After irritating the stomach end in B:14 days, get pancreas and be H﹠amp; E dyeing is illustrated as insulitis standards of grading (0 assigns to 4 fens).C: the insulitis of mice pancreatic scoring situation after berberine hydrochloride or the solvent administration.

The cytokine-expressing spectrum of the NOD mice that Fig. 2, berberine are handled.

The female NOD mouse stomach in 11 weeks gave berberine hydrochloride totally 14 days, took out spleen cell.α-CD3 antibody with variable concentrations stimulated 48 hours.Receive supernatant and measure cytokine IL-17 (A), IL-6 (B), IFN γ (C), IL-4 (D), IL-10 (E), TNF α (F) with the ELISA method.

Fig. 3, berberine have suppressed the differentiation of external Th17.

A: use the CD4+T cell that purification comes out from the NOD mice to cultivate 4 days under the Th17 differentiation condition, add the berberine hydrochloride of variable concentrations respectively, cell inner dyeing is observed the expression of IL-17 and IFN γ then.

B: above-mentioned CD4+T cell culture was checked IL-17 after 2 days, and the mRNA of IL-17F and ROR γ t expresses.

C: above-mentioned CD4+T cell adds illustrated various medicine respectively under above-mentioned condition of culture (concentration is respectively SB-202190 (0.5 μ g/ml), PD98059 (10 μ M), SP600125 (1 μ M), BADGE (1 μ M), rosiglitazone (1 μ M), wortmannin (1 μ M)) cultivated 4 days, cell inner dyeing is observed the expression of IL-17 and IFN γ then.

D: above-mentioned CD4+T cell adds illustrated various medicine (wherein the berberine hydrochloride salinity is 10 μ M) respectively under above-mentioned condition of culture cultivated 4 days, and cell inner dyeing is observed the expression of IL-17 and IFN γ then.

The E:CD4+T cell is cultivated under following condition of culture respectively: the antibody of α-CD3/28 (available from BDPharmingen) (with numeral " 1 " expression), Th17 condition of culture (with numeral " 2 " expression) or Th17 condition of culture add berberine hydrochloride (with numeral " 3 " expression).Check the background (Erk1/2) of Erk and the expression of phosphorylation (p-Erk1/2) with immunoblotting then.The optical density of band is illustrated in the illustration on the right.

The female NOD mouse stomach in F:11 week is given berberine hydrochloride (consumption is the 200mg/Kg body weight) totally 14 days, takes out spleen cell, checks the background of Erk and the expression of phosphorylation with immunoblotting.In the illustration on the right of the optical density of band is illustrated in (n=4, * *, p＜0.01).

Fig. 4, Erk are the differentiation negative regulatory factors of the Th17 of people and mice.

A: use the CD4+T cell that purification comes out from the NOD mice to cultivate 4 days under the Th17 differentiation condition, add the PD98059 of variable concentrations respectively, cell inner dyeing is observed the expression of IL-17 and IFN γ then.

B, C: with retroviral infection the CD4+ cell of Erk of wild type (B) or sudden change (C) under the Th17 differentiation condition, cultivate, the expression of IL-17 and IFN γ is observed in dyeing in the born of the same parents.

Continuous three days lumbar injection PD98059 of D:NOD mice, the CD4+T cell that purification comes out was cultivated 4 days under the Th17 differentiation condition, and cell inner dyeing is observed the expression of IL-17 and IFN γ then.

E: the CD4+T cell of separation of human was cultivated 3 days under the Th17 differentiation condition, added the medicine shown in the figure respectively, detected the secretion of IL-17 with ELISA.

Fig. 5, berberine have suppressed the differentiation of external Th1.

A: use the CD4+T cell that purification comes out from the NOD mice to cultivate 4 days under the Th1 differentiation condition, add the berberine hydrochloride of variable concentrations respectively, cell inner dyeing is observed the expression of IL-17 and IFN γ then.

B: above-mentioned CD4+ cell culture checked that the mRNA of IFN γ and T-bet expresses after 2 days.

C: above-mentioned CD4+ cell adds illustrated various medicine respectively under above-mentioned condition of culture (concentration is respectively SB-202190 (0.5 μ g/ml), PD98059 (10 μ M), SP600125 (1 μ M), BADGE (1 μ M), Rosiglitazone (1 μ M), Wortmannin (1 μ M)) cultivated 4 days, cell inner dyeing is observed the expression of IL-17 and IFN γ then.

The D:CD4+T cell is cultivated under following condition of culture respectively: the antibody of CD3/28 (with numeral " 1 " expression), Th1 condition of culture (with numeral " 2 " expression) or Th1 condition of culture add berberine hydrochloride (with numeral " 3 " expression).Check p38MAPK and the background (p38) of JNK and the expression of phosphorylation (p-p38) with immunoblotting then.The optical density of band is illustrated in the illustration on the right.

The female NOD mouse stomach in E:11 week takes out spleen cell to berberine hydrochloride totally 14 days, checks p38MAPK and the background of JNK and the expression of phosphorylation with immunoblotting.In the illustration on the right of the optical density of band is illustrated in (n=4, * *, p＜0.01).

Fig. 6, berberine are by ubiquitinization-proteasome path degraded STAT4.

A: use the CD4+T cell that purification comes out from the NOD mice to cultivate under the Th1 differentiation condition, add the medicine shown in the figure respectively, immunoblotting detects associated protein (STAT1/4 background and phosphorylation and actin) and expresses.B: use the CD4+T cell that purification comes out from the NOD mice to cultivate under the Th1 differentiation condition, add the medicine shown in the figure respectively, immunoblotting detects associated protein (STAT4 and Actin) and expresses.C: use the CD4+T cell that purification comes out from the NOD mice to cultivate under the Th1 differentiation condition, add the medicine shown in the figure respectively, immunoblotting detects associated protein (STAT1/4 background and phosphorylation and Actin) and expresses.D: use the CD4+T cell that purification comes out from the NOD mice under the Th1 differentiation condition, to cultivate, add the medicine shown in the figure respectively, use antibody (available from Cell signalling) the sedimentation cell pyrolysis liquid of STAT4 then, immunoblotting detects associated protein (STAT4, Ubiquitin Actin) expresses.

The sketch map of the mechanism of Th17 and Th1 differentiation in Fig. 7, the berberine inhibition type i diabetes.

The specific embodiment

The inventor is through long-term and extensive studies, discloses berberine first for improving or autoimmune diseasees such as treatment type i diabetes, insulitis have extremely excellent effect.But the generation of the generation of the described berberine inflammation-inhibiting factor, promotion anti-inflammatory factors; But described berberine is the Th17 differentiation and the Th1 differentiation of suppressor T cell also; Described berberine also can reduce the protein expression level of STAT4 or increase the ubiquitinization of STAT4, thereby realizes the effect of control autoimmune disease.

Berberine

Berberine (Berberine) is a kind of natural herbal effective medicinal ingredient that derives from, and is a kind of alkaloid.In the prior art, berberine is used for the treatment of diarrhoea always, and modern pharmacology studies confirm that berberine has effects such as significant heart failure resistance, arrhythmia, cholesterol reducing, resisting vascular smooth muscle propagation, antiplatelet, thereby unifies at cardiovascular system and also to have effect aspect the nervous system disease.The structural formula of berberine is:

In the present invention, described " berberine " can be the chemical compound shown in the formula (I) that exists of respective pure form, or purity also can be to contain the berberine of 1-90wt% or the extract of its pharmaceutically acceptable salt greater than the chemical compound shown in 85% the formula (I).

The method that the preparation method of berberine can adopt the state of the art personnel to be understood.Described berberine can extract in any plant that contains berberine or derivatives thereof (as salt or ester), and described plant includes but not limited to: Rhizoma Coptidis, Cortex Phellodendri, Radix Berberidis.Can be with the direct administration of the plant extract of berberine; Perhaps the plant extract of berberine can be purified and processes, make the preparation of the berberine of respective pure form or various forms of berberines after, be used for administration.

In addition, described berberine also can obtain by the mode of chemosynthesis, and this is that present this area can be realized fully.

In the present invention, the pharmaceutically acceptable salt that also comprises berberine.Described " pharmaceutically acceptable salt " is meant the salt that berberine and mineral acid or organic acid reaction generate.Preferably, the pharmaceutically acceptable salt of described berberine is the hydrochlorate of berberine (being called berberine hydrochloride again), and it has the structural formula shown in the formula (II):

The isomer of berberine, racemic modification, hydrate or precursor also are included among the present invention, as long as they have the function of the control autoimmune disease identical or approaching with berberine.Described " precursor " refers to after taking with suitable method, and the precursor of this chemical compound carries out metabolism or chemical reaction and is transformed into structural formula (I) or a kind of chemical compound (II) or its analog in mammalian body.

Described berberine or its isomer, racemic modification, pharmaceutically acceptable salt, hydrate or precursor can obtain by the mode of chemosynthesis.

Purposes

The invention provides the purposes of berberine, its pharmaceutically acceptable salt or derivant, be used to prepare the compositions of prevention or treatment mammal autoimmune disease.

Described autoimmune disease preferably is meant Th1 or the relevant class disease of Th17 cell; More preferably, described autoimmune disease is a type i diabetes.

In order to prove the such use of berberine, the inventor has adopted classical type i diabetes model (NOD mice) to be object of study, gives berberine with the mode of irritating stomach, observes the incidence of type i diabetes then.The inventor's result of study finds that berberine can reduce the sickness rate of type i diabetes significantly, reduces the secretion of inflammatory factors such as IL-17 and IFN γ, has obviously improved the insulitis symptom simultaneously.Because Th17 and Th1 are two groups of T cells that cause insulitis and type i diabetes, the inventor has studied the influence of berberine for the differentiation of these two groups of T cells.External T cell differentiation experiment shows that berberine can suppress the differentiation of Th17 and Th1.Discover that further berberine is respectively by activating Erk and suppressing p38MAPK and JNK suppresses the differentiation of Th17 and Th1.The inventor's experimental result is also pointed out, and Erk is a negative regulatory factor of the Th17 differentiation of people and mice.On the other hand, berberine suppresses the activity of STAT1 and STAT4 by p38/MAPK and JNK, and has passed through the ubiquitin protein enzyme body STAT4 that degrades, and then the differentiation of regulation and control Th1.In a word, berberine passes through the differentiation of active and then regulation and control Th1 and the Th17 of regulation and control MAPK, thereby has reduced the sickness rate of type i diabetes and improved the islets of langerhans inflammation.So berberine also can be used for the improvement and the treatment of other autoimmune disease relevant with the Th17 cell with Th1.

The mechanism of Th17 and Th1 differentiation is referring to the sketch map of Fig. 7 in the berberine inhibition type i diabetes.

Compositions

As used herein, term " compositions of the present invention " comprises pharmaceutical composition and dietary supplement (as Halth-care composition), as long as they contain berberine or its pharmaceutically acceptable salt or the derivant active component as prevention or treatment mammal autoimmune disease.

The invention provides a kind of pharmaceutical composition, contain: (a) berberine of effective dose or its pharmaceutically acceptable salt or derivatives thereof; And (b) pharmaceutically acceptable carrier or excipient.

Among the present invention, term " contains " the various compositions of expression and can be applied to together in mixture of the present invention or the compositions.Therefore, term " mainly by ... form " and " by ... composition " be included in during term " contains ".

Among the present invention, " pharmaceutically acceptable " composition is to be applicable to people and/or animal and not have the material that excessive bad side reaction (as toxicity, stimulation and allergy) promptly has rational benefit/risk ratio.

Among the present invention, " pharmaceutically acceptable carrier " is acceptable solvent, suspending agent or the excipient pharmaceutically or on the food that is used for berberine of the present invention or its physiologically acceptable salt or derivatives thereof are sent to the animal or human.Carrier can be a liquid or solid.

From being easy to prepare the position with administration, preferred pharmaceutical composition is a solid-state composition, and especially tablet and solid are filled or the capsule of liquid filling.The oral administration of pharmaceutical composition is preferred.

The dosage form of pharmaceutical composition of the present invention can be diversified, all is fine so long as can make active component arrive the intravital dosage form of mammal effectively.Such as being selected from: tablet, capsule, powder, granule, syrup, solution, suspension or aerosol.Wherein berberine may reside in suitable solid or liquid support or the diluent.

Berberine of the present invention and compositions thereof also can be stored in the disinfector that is suitable for injecting or instils.

Usually, in pharmaceutical composition of the present invention, berberine accounts for the 1-50% of gross weight as active component; 2-40% preferably; 3-30% more preferably, all the other are materials such as pharmaceutically acceptable carrier and other additives.

Usually, described dietary supplement contains berberine or its pharmaceutically acceptable salt or derivatives thereof of 0.001-50wt%; (b) acceptable carrier or excipient on the food.

Administering mode and dosage form

Pharmaceutical composition of the present invention can be made the dosage form of any routine by conventional method.

The effective dose of used active component can change with the order of severity of the pattern of used chemical compound, administration and disease to be treated.Yet, usually when berberine of the present invention or its pharmaceutically acceptable salt or derivatives thereof every day give with the dosage of about 1-300mg/kg the weight of animals, can obtain gratifying effect, preferably give with the dosage that separates for 1-3 time every day, or with the slow release form administration.For most of large mammal, the accumulated dose of every day is about 5-1000mg, preferably is about 10-500mg.Be applicable to dosage form for oral administration, comprise reactive compound with the intimately mixed about 1-200mg of solid-state or liquid pharmaceutically acceptable carrier.This dosage of scalable is replied so that optimal treatment to be provided.For example, by an urgent demand of treatment situation, but give the dosage that several times separate every day, or dosage is reduced pari passu.

Described chemical compound or its pharmaceutically acceptable salt and compositions thereof can be by oral and intravenous, intramuscular or administrations such as subcutaneous; Oral administration preferably.Solid-state carrier comprises: starch, lactose, dicalcium phosphate, microcrystalline Cellulose, sucrose and kaolin, and liquid carrier comprises: sterilized water, Polyethylene Glycol, nonionic surfactant and edible oil (as Semen Maydis oil, Oleum Arachidis hypogaeae semen and Oleum sesami), as long as be fit to the characteristic of active component and required specific administration mode.Normally used adjuvant also can advantageously be comprised in pharmaceutical compositions, for example flavoring agent, pigment, antiseptic and antioxidant such as vitamin E, vitamin C, BHT and BHA.

The medicament forms that is adapted to inject comprises: aseptic aqueous solution or dispersion liquid and aseptic powder (being used for preparing aseptic injectable solution or dispersion liquid) temporarily.In all situations, these forms must be aseptic and must be that fluid is discharged fluid to be easy to syringe.Under manufacturing and condition of storage must be stable, and must be able to prevent the pollution effect of microorganism (as antibacterial and fungus).Carrier can be solvent or disperse medium, wherein contains just like water, alcohol (as glycerol, propylene glycol and liquid polyethylene glycol), their suitable mixture and vegetable oil.

Berberine or its pharmaceutically acceptable salt and compositions thereof also can with other active component or medication combined administration.

When two or more medication combined administration, generally have and be better than the individually dosed respectively effects of two kinds of medicines.

Major advantage of the present invention is:

(1) found the new purposes of berberine in autoimmune diseasees such as control type i diabetes first, its pharmacological action is strong, and does not have toxic and side effects, has fabulous prospect in medicine.

(2) described berberine can directly extract in plant, and is low in toxicity, in the time of taking convenience, also has the source and enriches, cheap, the simple advantage of preparation technology.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise percentage ratio and umber calculate by weight.

Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.

Embodiment 1, berberine have reduced the onset diabetes rate of diabetic mice

(structural formula is suc as formula shown in (II), available from Sigma) is formulated in solvent PBS with berberine hydrochloride, forms uniform turbid liquid, is used for administration type i diabetes mice (NOD mice).

Can the inventor has detected berberine reduce the onset diabetes rate of NOD mice (available from this Leco Corp. of Shanghai City).The female NOD mouse stomach in 11 weeks is to berberine hydrochloride totally 14 days (giving berberine hydrochloride 200mg/Kg body weight every day).Measure random blood sugar weekly, can be regarded as diabetes when blood glucose is higher than 250mg/dL, observe the mice number that diabetes take place.And, after irritating the stomach end in 14 days, get pancreas and be H﹠amp; E dyeing, a situation arises to judge insulitis to observe the dyeing situation.With solvent administration group NOD mice in contrast.As a result, 100% matched group NOD mouse invasion, but the berberine hydrochloride processed group has only 50% mouse invasion, sees Figure 1A.

The insulitis of the mice of berberine hydrochloride processed group has also obtained alleviation.The standards of grading of insulitis have represented in Figure 1B.The inventor's analysis shows that after the berberine hydrochloride administration, insulitis does not take place 77.0% islets of langerhans; This numeral is 45.2% at matched group, sees Fig. 1 C.In addition, generally speaking, the insulitis of the mice of berberine hydrochloride processed group has also obtained significant reduction.

The above results represents, berberine is for the significant therapeutical effect that has of the diabetes of NOD mice.

Embodiment 2, berberine are handled the cytokine-expressing spectrum that has changed the NOD mice significantly

Next the inventor has analyzed the secretion situation that Rhizoma Coptidis have the cytokine that does not change mice.

The female NOD mouse stomach in 11 weeks is to berberine hydrochloride totally 14 days (giving the berberine hydrochloride of 200mg/Kg body weight every day), with solvent administration group NOD mice in contrast.Conventional method is separated the spleen cell of mice, (antibody (available from BD Pharmingen) of α-CD3) was handled 48 hours with anti-CD3, receive supernatant, detect the concentration of IL-17, IL-6, IFN γ, TNF α, IL-4 and IL-10 with conventional enzyme-linked immunosorbent assay (ELISA) respectively.With solvent administration group NOD mice in contrast.

Found that inflammatory factors such as IL-17, IL-6, IFN γ and TNF α descend, anti-inflammatory factors IL-4 and IL-10 then have rising in various degree, see Fig. 2.

Embodiment 3, berberine are by activating the vitro differentiation that Erk has suppressed Th17

Because IL-17 is the labelled molecule of Th17,, the inventor may influence to some extent for the differentiation of Th17 so inferring berberine.

The CD4+T cell that comes out with purification from the NOD mice under the Th17 differentiation condition (at IL-6[10ng/mL], TGF β [2ng/mL], IL-1 β [10ng/mL], IL-23[10ng/mL], α-IL-4 antibody [10 μ g/mL] is hatched under α-IFN gamma antibodies [the 10 μ g/mL] condition) cultivated 4 days, add variable concentrations (5 μ M respectively, 10 μ M) berberine hydrochloride, cell inner dyeing is observed the expression of IL-17 and IFN γ then.As a result, do not give under the situation of berberine the cell that about 20% T cell has become secretion IL-17.But give the percentage ratio of the inhibition IL-17 positive cell that berberine hydrochloride can concentration relies on, see Fig. 3 A.

Except the secretion of using FACS technology for detection IL-17, the inventor also adopts conventional RealTime round pcr to detect several vital signs molecules of Th17 differentiation, comprise IL-17, IL-17F (IL-17F is the cytokine with the same family of IL-17, also is the significant labelled molecule of Th17) and Ror γ t.Behind the CD4+ cell culture 2 days, check IL-17, the mRNA of IL-17F and ROR γ t expresses, and (wherein α-CD3/28 represents α-CD3/28 antibody incubation cell, and α-CD3/28 antibody is that the growth of T cell is necessary to the results are shown in Figure 3B; Cocktail represents IL-6[10ng/mL], TGF β [2ng/mL], IL-1 β [10ng/mL], IL-23[10ng/mL], α-IL-4 antibody [10 μ g/mL], α-IFN gamma antibodies [10 μ g/mL] incubated cell).These expression of gene all use actin (actin) to carry out standardization as confidential reference items.

In order to understand that berberine is the differentiation what mechanism to suppress Th17 by, the inventor used the agonist or the inhibitor of a series of signal path.These signal paths are all confirmed it is to be subjected to the berberine regulation and control by other laboratorys.These agonist and inhibitor comprise: the SB-202190 (inhibitor of p38MAPK, available from Sigma), PD98059 (inhibitor of MEK1/2-Erk1/2 is available from Sigma), the SP600125 (inhibitor of JNK/SAPK, available from Sigma), BADGE (inhibitor of PPAR α is available from Sigma), the Rosiglitazone (agonist of PPAR γ, available from Sigma) and Wortmannin (inhibitor of PI3K/Akt is available from Sigma).In addition, the inventor has also used Compound C (inhibitor of AMPK is available from Sigma), but in experiment, Compound C has presented a kind of broad-spectrum and suppressed and cytotoxicity, so the inventor has just abandoned the research to Compound C.Very fortunately, the inventor has found that PD98059 can raise the differentiation of Th17, sees Fig. 3 C.

In addition, PD98059 can also reverse the inhibition of the differentiation of berberine hydrochloride salt pair Th17, sees Fig. 3 D.

In addition, the inventor finds that berberine hydrochloride all can activate Erk1/2 with external in vivo, sees Fig. 3 E, F.

The above-mentioned Notes of Key Data, berberine hydrochloride may suppress the Th17 differentiation by activating Erk.

Embodiment 4, Erk are to the regulation and control of Th17 differentiation

The inventor's The above results prompting: Erk probably is the negative molecule of regulating of in the Th17 differentiation; In order to verify this hypothesis, the inventor at first detects the effect of PD98059 in the Th17 differentiation.Cultivated 4 days under the Th17 differentiation condition with the CD4+T cell that purification from the NOD mice comes out, add variable concentrations (10 μ M respectively, 20 μ M) PD98059, cell inner dyeing is observed the expression of IL-17 and IFN γ then, with the cell that do not add PD98059 (Control) in contrast.The result shown in Fig. 4 A, the differentiation of the promotion Th17 that PD98059 can concentration relies on.

With wild type Erk gene (Erk1:gene ID 5595; Erk2:gene ID 26413) and mutated genes (on the protein level Erk1 and Erk2 the 71st and the 52nd lysine are become the encoding gene that constitutes behind the arginine; Available from U.S. Southwestern Medical Center) be cloned in the BamHI/XhoI restriction enzyme site of pM-IRES-GFP carrier (available from U.S. CellBiolab company), constitute recombinant expression carrier, utilize to be used for the cell Plat-E (U.S. Cell Biolab company) of packaging virus specially and to carry out virus packing, the retrovirus of the Erk of wild type or sudden change is carried in acquisition.

With retroviral infection the CD4+ cell of Erk of wild type or saltant under the Th17 differentiation condition, cultivate, the expression of IL-17 and IFN γ is observed in dyeing in the born of the same parents.The result is shown in Fig. 4 B and 4C, and the Erk of mutant form can promote the differentiation of Th17.

In order further to detect the effect of Erk in the Th17 differentiation, the inventor gives injected in mice PD98059 (for three days on end, every day 1 time, dosage as shown in the figure).After 3 days, take out the T cell of mice and do the Th17 differentiation, found that the processing of PD98059 can improve the ability of T cell to the Th17 differentiation, sees Fig. 4 D.

In addition, the inventor also has more significant discovery a: Erk that the negative regulation effect of the differentiation of Th17 be can be generalized to people's T cell, sees Fig. 4 E.

Embodiment 5, berberine are by suppressing the differentiation that p38MAPK and JNK suppress Th1

Well-known, Th1 is the important virulence factor that type i diabetes takes place.Because can the inventor's preliminary data show berberine and can reduce the secretion of the IFN γ of mice, influence the differentiation of Th1 so the inventor is concerned about berberine.

Shown in Fig. 5 A, under the Th1 differentiation condition, to cultivate 4 days, about 80% T cell has become the cell of secretion IFN γ.But the percentage ratio of the inhibition IFN γ positive cell that berberine hydrochloride can concentration relies on.

Except the secretion of using FACS technology for detection IFN γ, the inventor has also detected several vital signs molecules of Th1 differentiation with Real Time round pcr, comprise IFN γ and T-bet, these expression of gene are all done confidential reference items with actin and have been carried out standardization, the results are shown in Figure 5B.

In order to understand that berberine hydrochloride is the differentiation what mechanism to suppress Th1 by, the inventor continues to use the agonist or the inhibitor of above-mentioned signal path.Very fortunately, the inventor has found the differentiation of SB-202190 and SP600125 downward modulation Th1, sees Fig. 5 C.

In addition, the inventor find berberine hydrochloride in vivo with the external activity that all can reduce p38 and JNK (ratio that phosphorylation promptly takes place descends), see Fig. 5 D, E.

These Notes of Key Datas, berberine hydrochloride are to suppress the differentiation of Th1 by the activity that suppresses p38 and JNK.

Embodiment 6, berberine reduce the protein expression level of STAT4 by Ubiquitin-Proteasome Pathway

Because STAT1 (downstream molecules of IFN γ) and STAT4 (downstream molecules of IL-12) play the part of important effect in the differentiation of Th1, so the inventor has also detected the background of berberine hydrochloride salt pair STAT1 and STAT4 and the expression of phosphorylation.As shown in Figure 6A, berberine hydrochloride has significantly reduced the phosphorylation level of STAT1 and STAT4.

Very ironically, the inventor also has a unexpected discovery: berberine hydrochloride can also reduce the stability of STAT4 except the phosphorylation level that has reduced STAT4.If add MG132 (proteasome inhibitor is available from Cell signalling), the reduction of berberine hydrochloride salt pair STAT4 background just has been suppressed, and sees Fig. 6 B.

Whether because find in the superincumbent test of the inventor that berberine hydrochloride can reduce the activity of p38 and JNK,, the inventor changes the expression of STAT4 by p38 and JNK so wondering berberine hydrochloride.The result shows that the inhibitor of p38 and JNK can not change the background of STAT1 and STAT4 expresses; Can only change the phosphorylation of STAT1 and STAT4, see Fig. 6 C.

For further checking berberine hydrochloride by the uiquitin-protease enzyme body STAT4 that degrades, the inventor is hatched with antibody and the cell pyrolysis liquid of STAT4, use proteinA/G agarose beads sedimentation then, detect ubiquitin with Western blot at last.Found that berberine hydrochloride can significantly increase the ubiquitinization of STAT4, sees Fig. 6 D.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Claims (10)

1. the purposes of berberine, its pharmaceutically acceptable salt or derivatives thereof is characterized in that, is used to prepare the compositions of prevention or treatment autoimmune disease.

2. purposes as claimed in claim 1 is characterized in that, the pharmaceutically acceptable salt of described berberine is the hydrochlorate of berberine.

3. purposes as claimed in claim 1 is characterized in that, described autoimmune disease is Th1 or the relevant autoimmune disease of Th17 cell.

4. purposes as claimed in claim 1 is characterized in that described autoimmune disease is a type i diabetes.

5. purposes as claimed in claim 1 is characterized in that, described compositions also is used for: prevention or treatment insulitis.

6. purposes as claimed in claim 1 is characterized in that, described compositions also is used for: the generation of the inflammation-inhibiting factor; Or the generation of promotion anti-inflammatory factors.

7. purposes as claimed in claim 6 is characterized in that, described inflammatory factor is selected from: IL-17, IL-6, IFN γ or TNF α; Or

Described anti-inflammatory factors is selected from: IL-4 or IL-10.

8. purposes as claimed in claim 1 is characterized in that, described compositions also is used for: the Th17 differentiation of suppressor T cell.

9. purposes as claimed in claim 1 is characterized in that, described compositions also is used for: the Th1 differentiation of suppressor T cell.

10. purposes as claimed in claim 1 is characterized in that, described compositions also is used for: the protein expression level that reduces STAT4; Or increase the ubiquitinization of STAT4.

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