CN102793712A - Application of chrysin in preparation of medicaments for treating autoimmune and inflammatory diseases - Google Patents

Abstract

The invention discloses application of chrysin in preparation of medicaments for treating autoimmune diseases. Experimental results show that the chrysin can inhibit differentiation of human immune dendritic cells (DC) and maturity of LPS (lipopolysaccharide)-activated human immune DC, can inhibit the antigen uptake capabilities of the immature DC and the antigen presenting capabilities of the mature DC and can inhibit T cell proliferation reactions stimulated by the DC. In particular, the chrysin can reduce the average score, the highest score and the total score of clinical scores of experimental autoimmune encephalomyelitis of mice and can relieve myelitis cell infiltration and demyelination in spinal cords. Thus, the medicament inhibiting immune systems is expected to become the medicament for treating such autoimmune inflammatory diseases as multiple sclerosis, neuromyelitis optica and acute disseminated encephalomyelitis clinically.

Description

The application of chrysin aspect preparation treatment autoimmune and diseases associated with inflammation medicine

Technical field

The invention belongs to the Chinese medicine applied technical field, relate to Chinese medicine monomer chrysin (Chrysin) and aspect treatment autoimmune disease, nervous system inflammation property disease medicament, use in preparation.

Background technology

Flavone compound is the one type of important natural organic-compound that produces in the vegetable metabolic process, and treatment of diseases such as the mankind's tumor, aging, cardiovascular and prevention are had great significance.Flavone compound is the focus that research and development both at home and abroad utilize always over particularly past 30 years, is to seek the lead compound of development prospect and the source of biological activity are arranged.Chrysin (Chrysin) promptly 5,7. dihydroxyflavone is a kind of natural flavone compounds, extensively is present in various plants, propolis and the Mel, is the main effective ingredient of propolis.Research shows that chrysin has multiple pharmacologically actives such as antioxidation, anti-inflammatory, antiviral, resisting hypertension, blood sugar lowering, estrogen antagonist, anxiety, antiallergic and inhibition aromatase activity.The antitumor action of chrysin mainly comprises: anti-tumour cell proliferative; Inducing apoptosis of tumour cell; The reversing multiple medicine resistance of tumor cells effect; Anti-gene mutation effect etc.But chrysin is very few to the influence report of autoimmune disease and nervous system inflammation property disease.

In the immune defence reaction of body; BMDC (dendritic cells; DC) find in 1973 by American scholar Steinman, be know at present function the strongest and unique antigen presenting cell that can activate primary tape T cell (antigen presenting cell, APC).(dendritic cells DC) is distributed widely in brain body tissue and internal organs in addition to dendritic cell, and negligible amounts only accounts for 1% of human peripheral blood single nucleus cell, and so it has the dendritic projection of a lot of branches name.According to the source, can DC be divided into two types, promptly derive from the marrow appearance BMDC and the lymph appearance BMDC that derives from LSC of myeloid stem cell.BMDC is full-time antigen presenting cell, and its major function is picked-up, processed and offers antigen, starts specific immune response.Immature DC can be expressed MHC I/class (people's MHC is called HLA), IgG Fc receptor, C3b receptor and some Toll appearance receptor.Immature DC is absorbed, the processed antigenic capacity is strong, and offers a little less than the antigen stimulation immunne response ability.Ripe DC surface character property flag bit CD1a, CD11c and CD83, but high expressed MHC II/class and costimulatory molecules (like CD80, CD86, a little less than its picked-up, the processed antigenic capacity, and offer antigen, startup immunne response ability is strong.DC can induce the T cells activation, therefore is the moving person of beginning of body specific immune response.BMDC also is an immunity regulatory cell important in the body, can participate in intrinsic and adaptive immune response through the secretion different cells factor.For example: it is main cytokine that some DC can secrete IL-12, induces or promotes T cells to be divided into the Th1 cell, strengthens cellullar immunologic response; It is main cytokine that some DC can secrete with the interferon type, produces effects such as infection and immunomodulating; In some cases, DC can induce the B cell that the conversion of IgG classification takes place through cytokines such as secretion IL-10 and TGF-β, produces the IgA antibody-like; Can be master's cytokine with IL-1 β also, promote T, B cell activation through secretion.APC comprises macrophage, B cell, BMDC etc.Wherein DC knows the strongest full-time APC of function in the body at present; Stretch out many pseudopodium appearance or dendron appearance projection when its common trait is promptly ripe as stated; Express MHC-

, II class antigen, costimulatory molecules such as high expressed CD80, CD86 and express ripe surface markers CD83; Offer antigen, to start the immunne response ability strong.Different with other APC, DC can induce primary tape T cell proliferation, and macrophage and B cell can only stimulate activatory or memory t cell, so DC has unique status in immunne response.Meanwhile, the effect of DC in autoimmune disease takes place also more and more paid attention to by people.DC all can influence the final result of experimental autoimmune disease to antigenic transmission and to the turnover of the antigenic peptides of offering.Experiment shows: the antigen (comprising the process of angtigen presentation being given the T cell) that DC is transmitted carries out the selectivity intervention, or all can prevent and treat the generation of autoimmune disease with the DC inducing immune tolerance.

Multiple sclerosis (Multiple Sclerosis) is to become characteristics with central nervous system's white matter demyelinating disease, good sending out in person between twenty and fifty, and age of onset is many to have the characteristics of high disability rate, high recurrent at 20～40 years old, and the part conditions of patients is carrying out property to be increased the weight of.Still there is not effective healing way certainly so far.EAE (EAE; Experimental autoimmune encephalomyelitis) is the mature animal model of multiple sclerosis (MS), because of its pathology generation and the clinical manifestation that can simulate multiple sclerosis is widely used.Mostly laboratory sensitinogen commonly used is brain or myeloid tissue's homogenate, MBP composition or its polypeptide fragment etc.; Mostly the immunity object is that rodent makes up the EAE model; Different sensitinogens can produce the different EAE model of disease symptom, can be according to the type selecting of its EAE model that will set up different sensitinogens and the different immune objects of quilt.This experiment selects for use myelin oligodendrocyte glycoprotein (MOG) to make MOG as antigen immune C57BL/6 mice _35-55-EAE mouse model.Though content is seldom in myelin protein for the myelin oligodendrocyte glycoprotein of using in this experiment; Only account for the 0.01%-0.05% of myelin protein composition; But because MOG is present in the outermost layer of myelin film and oligodendrocyte; Have hyperimmunization originality, and in making up the EAE process, find that MOG is that unique demyelination antibody response that can cause can cause central nervous system's myelin protein composition of t cell responses again.Therefore, go immune mouse can set up pathogeny and clinical symptoms thereof with myelin oligodendrocyte glycoprotein with the extremely similar EAE model of MS.At present, there are some researches show that MOG and anti-MOG antibody play a part very important in the pathogenic process of MS.The domestic and international in recent years related experimental methods of this laboratory reference is set up the inductive stable chronic EAE ideal model of MOG.

At present; The pathology mechanism of EAE/MS is not also understood fully; But generally believe that EAE is the inflammatory disease of the central nervous system that is caused by the autoimmune inflammation that helper T lymphocyte (TH1 and TH17) mediates, the directed differentiation of different helper T lymphocytes depends on that the different antigen signals in BMDC source stimulate.DC contacts external or autoantigen, and these antigens are digested classification, corresponding immune signal is passs T cell and B cell then, again through different T cells and the B cell completion body's immunological function of sharing out the work and help one another.The T cell be in the immunocyte than large group, be divided into different subgroups (as CD4 positive with the CD8 positive two large groups).The positive T cells of CD4 (Na ve T cell) stimulates through the unlike signal (like different cytokines) that derives from BMDC, and then directed differentiation is that different T cell subsets is exercised different immunologic functions again.The CD4 positive T cell is divided into helper T lymphocyte and regulatory T cells again.Helper T lymphocyte is divided into TH1, TH2, TH17 three large groups.TH1 stimulates the positive T cells of CD4 by IL-12 or IFN-γ, differentiates TH1 emiocytosis IFN-γ, but the initial and inflammatory reaction of mediated cell immunity through Transcription Factor T-bet.Research shows the unbalance generation that causes autoimmune disease in the IFN-γ body; The TH2 cell stimulates the positive T cells cell of CD4 by IL-4, and GATA-3 differentiates through transcription factor, and it mainly suppresses inflammatory reaction through anti-inflammatory factors such as secretion IL-4 ﹑ IL-10 in body.Therefore, suppress the release of inflammatory factor and the secretion of promotion anti-inflammatory factors and become one of direction of treatment autoimmune disease.

Summary of the invention

The objective of the invention is to disclose the application of a kind of chrysin (Chrysin) aspect preparation conduct treatment LADA, nervous system inflammation property disease medicament.Wherein the effective dose of chrysin in treatment autoimmune disease, nervous system inflammation property disease medicament is preferably 75 mg/Kg body weight/day.

The present invention further discloses chrysin and suppress the application in the immunologic rejection medicine as the treatment organ transplantation in preparation.Experimental result shows that chrysin (Chrysin) suppresses the maturation figure (flow cytometer datagram) of the activated people's dendritic cell of LPS; It can suppress immune activation, therefore can treat autoimmune disease or suppress the immunologic rejection in the organ transplantation.

Autoimmune disease of the present invention refers to: the autoimmune disease that immune BMDC or T cell are relevant.

The autoimmune disease that immune BMDC of the present invention or T cell are relevant refers to: multiple sclerosis (Fig. 1-11); Rheumatoid arthritis (Fig. 1-6); LADA retinitis (Fig. 1-6); Optic neuromyelitis (Fig. 1-6); Systemic lupus erythematosus (sle) (Fig. 1-6); Struma lymphomatosa (Fig. 1-6); Autoimmune hemolytic anemia (Fig. 1-6); AT (Fig. 1-6); Myasthenia gravis (Fig. 1-6); Primary biliary cirrhosis (Fig. 1-6); Aggressive chronic hepatitis (Fig. 1-6); Chronic glomerulus scorching (Fig. 1-6); Myositis (Fig. 1-6); Systemic sclerosis (Fig. 1-6).

Nervous system inflammation property disease of the present invention refers to multiple sclerosis (Fig. 1-11), optic neuromyelitis (Fig. 1-6) and acute disseminated encephalomyelitis (Fig. 1-11); Its effective dose is preferably 75 mg/Kg body weight/day.The taking dose of amounting to into the adult was according to body weight 50-60Kg:3.75g-4.5g/ days.

The present invention uses immunocyte differentiation and activation model and experimental animal model to carry out a large amount of pharmacological evaluation, and experimental result shows: chrysin can suppress the differentiation of people's immunity BMDC, suppresses the Maturity of the activated people's immunity of LPS BMDC; The antigen uptake ability of immaturity dendritic cell and the angtigen presentation ability of mature dendritic cell can be suppressed, the T cell proliferative response that dendritic cell stimulate can be suppressed.Particularly chrysin can reduce mouse experiment systemic autoimmune encephalomyelitis clinical score average mark, clinical score best result and clinical score total points; Can alleviate spinal cord inflammatory cell infiltration and spinal cord demyelination.Experimental autoimmune encephalomyelitis is the experimental animal model of multiple sclerosis, optic neuromyelitis and acute disseminated encephalomyelitis, also is the typical animal model of research autoimmune and diseases associated with inflammation.Therefore, the present invention is expected to become clinically the medicine of autoimmune inflammation property diseases such as treatment multiple sclerosis and acute disseminated encephalomyelitis.

Immunologically competent cell such as BMDC etc. have the identification exotic antigen or think mechanism to the deleterious autoantigen of organism.Immunocyte is discerned autoantigen and is caused that autoimmune response is the pathogenesis basis of panimmunity disease.For example multiple sclerosis, rheumatoid arthritis, LADA retinitis, optic neuromyelitis, systemic lupus erythematosus (sle), struma lymphomatosa, autoimmune hemolytic anemia, AT, myasthenia gravis, primary biliary cirrhosis, aggressive chronic hepatitis, chronic glomerulus inflammation, myositis, systemic sclerosis etc.And the inhibition immunocyte, the material that especially suppresses the BMDC with professional antigen presentation ability in immune system forward position will have the purposes of potential treatment autoimmune disease.

Application of the present invention; Wherein autoimmune disease refers to the autoimmune disease of above-mentioned indication; Especially inflammatory disease of the central nervous system, for example people's multiple sclerosis, optic neuromyelitis and acute disseminated encephalomyelitis or the like are typically people's multiple sclerosis.

Description of drawings:

Fig. 1 is that chrysin (Chrysin) suppresses people CD14 ⁺Mononuclear cell is to the figure of dendritic cell differentiation (flow cytometer datagram);

Fig. 2 is that chrysin (Chrysin) suppresses people CD14 ⁺The figure (block diagrams of corresponding flow cytometer data) of monocytic dendritic cells differentiation;

Fig. 3 is that the antigen uptake of chrysin (Chrysin) inhibition immaturity dendritic cell can be tried hard to; It can suppress immune system to antigenic respond.In autoimmune disease, can reduce the ability of autoimmune cell identification autoantigen.Therefore can treat autoimmune disease;

Fig. 4 is the maturation figure (flow cytometer datagram) that chrysin (Chrysin) suppresses the activated people's dendritic cell of LPS; It can suppress immune activation, therefore can treat autoimmune disease or suppress the immunologic rejection in the organ transplantation;

Fig. 5 is the maturation figure (corresponding flow cytometer datagram) that chrysin (Chrysin) suppresses the activated people's dendritic cell of LPS; It can suppress immune activation, therefore can treat autoimmune disease or suppress the immunologic rejection in the organ transplantation;

Fig. 6 is that chrysin (Chrysin) suppresses allosome T cell proliferative response (CFSE method) figure that dendritic cell stimulate; It can treat the cell-mediated autoimmune diseasees of T such as multiple sclerosis;

Fig. 7 is that (Chrysin 75mg/kg) alleviates the occurring degree figure of mouse experiment systemic autoimmune encephalomyelitis to oral chrysin; Explain that chrysin can treat nervous system inflammation property diseases such as autoimmune disease, particularly multiple sclerosis, optic neuromyelitis and acute disseminated encephalomyelitis;

Fig. 8 is that (Chrysin 75mg/kg) alleviates the highest score figure of the morbidity clinical score of mouse experiment systemic autoimmune encephalomyelitis to chrysin; Explain that chrysin can treat nervous system inflammation property diseases such as autoimmune disease, particularly multiple sclerosis, optic neuromyelitis and acute disseminated encephalomyelitis;

Fig. 9 is that (Chrysin 75mg/kg) alleviates the gross score figure of the morbidity clinical score of mouse experiment systemic autoimmune encephalomyelitis to chrysin.Explain that chrysin can treat nervous system inflammation property diseases such as autoimmune disease, particularly multiple sclerosis, optic neuromyelitis and acute disseminated encephalomyelitis;

Figure 10 is that (Chrysin 75mg/kg) alleviates the spinal cord inflammatory cell infiltration figure (H&E dyeing) of mouse experiment systemic autoimmune encephalomyelitis to oral chrysin; Explain that chrysin suppresses the infiltration of central nervous system's inflammatory cell, can treat central nervous system's autoimmune diseases associated with inflammation especially multiple sclerosis, optic neuromyelitis and acute disseminated encephalomyelitis;

Figure 11 is that (Chrysin 75mg/kg) alleviates the spinal cord demyelination (Luxol fast blue dyeing) of mouse experiment systemic autoimmune encephalomyelitis to oral chrysin; Explain that chrysin can treat disease multiple sclerosis, optic neuromyelitis and the acute disseminated encephalomyelitis of the characteristics that become with central nervous system's white matter demyelinating disease; (n=8, figure is expressed as mean+SD, * p 0.05, * * p 0.01, * * * p 0.001, the t check).

The specific embodiment:

Below in conjunction with embodiment the present invention is described, the scheme of embodiment described here does not limit the present invention; One of skill in the art can make improvements and change according to spirit of the present invention; Described these improvement and variation all should be regarded as within the scope of the invention, and scope of the present invention and essence are limited claim, and wherein chrysin (Chrysin) has commercially available; The reagent that other is used all has commercially available except that special mark.Wherein blood need pass through the approval of administration of health department.

Embodiment 1

1. 1 human peripheral blood mononuclear cell's separation

(1) fresh blood (being no more than 8 hours) is got at the station of dehematizing.Be approximately 40 mL.

(2) PBS with 3 times of volumes dilutes (blood sample of dilution is many more, and monocytic purification is good more), totally 160 mL.The lymphocyte separation medium Ficoll-Paque that gets 15 mL adds the centrifuge tube of 50 mL, and centrifuge tube inclination 45o draws the blood of 35 mL dilution, and 1 cm place is superimposed upon on the separating medium along tube wall at the separation liquid level, does not destroy separating interface.(divide 4 pipes, in the lymphocyte separation medium Ficoll-Paque of every pipe 15 mL, add 35 mL blood, the blood of average residual more earlier)

(3) centrifugal 30 min of room temperature 400g slowly slow down, and the brake shelves can not be set.

(4) steadily take out centrifuge tube, orlop is erythrocyte and granulocyte, and the intermediate layer is a lymphocyte separation medium, and the superiors are blood plasma and diluent etc.Muddy the sucking-off upper strata stays mononuclear cell cellular layer (one deck tunica albuginea) to greyish white layer (tunica albuginea) in order to be prone to for plasma layer and separating medium intersection, be distributed in practise physiognomy on.

(5) carefully shift tunica albuginea (lymphocyte, mononuclear cell, platelet) to new 50mL centrifuge tube.Be transferred to two pipes.

(6) phosphate buffer (PBS) of 3 times of volumes of adding in centrifuge tube mixes centrifugal 10 min of room temperature 300g.Carefully remove supernatant fully.

(7) in order to remove platelet, re-suspended cell precipitates in the buffer of 50 mL, and centrifugal 15 min of 200g carefully remove supernatant fully under the room temperature.When 200g was centrifugal, the platelet major part rested in the supernatant.

(8) count behind the PBS mixing of 3 times of volumes of adding.

(9) repeat again to wash 1 time.

Annotate: PMBC possibly be stored in 5% bovine serum albumin (BSA) or serum and the anticoagulant (heparin, EDTA or citric acid phosphoric acid glucose) and spend the night.Cell is stored in refrigerator and is no more than one day.

1.2 human CD14 ⁺Monocytic purification

(1) gets the cell suspending liquid of 80 μ L and the CD14 of 20 μ L ⁺Hatch 15 min for 4 ℃ behind microballon (available from the beautiful day Ni company) mixing, (cell and microballon are pressed 4:1 and added) will be mixed once in the centre.(all hemocytees add 300 μ L magnetic beads, add the PBS that 1200 μ L contain 0.5%BSA again)

(2) centrifugal 5 min of PBS (containing 0.5%BSA) 200g with 20 times of volumes (2mL) pre-cooling wash once, abandon supernatant, add the PBS of 500 μ L.

(3) during centrifugal, discrete magnets be installed on the multipurpose bracket, and the sorting post is placed in the magnet, the sorting post is transferred a clean aseptic centrifuge tube.With meeting cold phosphate buffer flushing sorting post 3 times, each 0.5 mL.Abandon eluent, again at clean aseptic centrifuge tube of sorting post underlying, with on the cell suspension appearance to the sorting post.

(4) with PBS buffer flushing detached dowel, each 0.5mL washes 3 times (first 0.5 mL adds slowly in order to avoid the cell in the disturbance sorting post).

(5) treat that the cell suspension all-pass is crossed post after, take off the sorting post from magnet, be placed on the aseptic 15 mL core barrels.In the sorting post, add the full RPMI1640 culture medium of 1 mL, piston is filled in detached dowel and eluting CD14 ⁺Mononuclear cell, counting.With CD14 ^-Cell cryopreservation is used for the T cell proliferation experiment.

(6) flow cell sorter is confirmed purity (95%)

(7) with containing 1000 U/mL GM-CSF (granulocyte macrophage colony stimulating factor); The complete 1640 culture medium (Sodium Pyruvate that contains 10 mM of 1000 U/mL IL-4 (interleukin 4); The glutamine of 10 mM, 100 μ g/mL kanamycin) the adjustment cell concentration is 1X10 ⁶Individual/mL also adds medicine.Cell suspending liquid 2 mL/ holes are inoculated in 12 orifice plates.

1.3. chrysin (Chrysin) processing time and concentration are confirmed

The BMDC that obtains (DC) is divided into two groups, and according to consulting document, every group has been carried out various concentrations over control treatment:

First group (differentiation): dosing in first day, carried out Flow cytometry on the 5th day.Chrysin handles different concentration:

the normal control group;

5 μ M/L Chrysin handle five days groups; 25 μ M/L Chrysin handle five days groups.

Second group (maturation): (lipopolysaccharide, lipopolysaccharide) (1 μ g/ml) carried out Flow cytometry on the 7th day for dosing in the 5th day and LPS.Chrysin handles different concentration: negative control group (Chrysin and LPS do not add);

matched group (only adding LPS);

5 μ M/L chrysins (Chrysin)+LPS handles two days groups;

25 μ M/L Chrysin+LPS handle two days groups.

At the 3rd day that cultivates, draw 50% supernatant, add the GM-CSF that contains 1000 U/ml (granulocyte macrophage colony stimulating factor) of equivalent, 1640 culture medium of the IL-4 of 1000 U/ml also add be used as medicine (adding by 2ml).Annotate: at the 5th day, in the hole of not dosing, add LPS and medicine respectively, and, continue in 1640 culture medium of the IL-4 of 1000 U/mL to cultivate two days at the GM-CSF of 1000 U/mL that contain equivalent (granulocyte macrophage colony stimulating factor).

Conclusion: chrysin (Chrysin) can suppress the differentiation (Fig. 1,2) of people's dendritic cell, the maturation (Fig. 4,5) of the activated people's dendritic cell of inhibition LPS, shows among the figure that the concentration along with chrysin increases, and the inhibition degree strengthens; The differentiation that suppresses dendritic cell can reduce the quantity of immunocyte, thereby reduces immunoreation; It can suppress immune activation simultaneously.In autoimmune disease, suppress the ability that immunoreation can alleviate autoimmune system attack autogenous cell or tissue.Therefore can treat autoimmune disease or suppress the immunologic rejection in the organ transplantation;

1.4 flow cytometer showed

(1) at sophisticated the 5th day, the 7th day last collecting cell of cell differentiation to the 1.5mL centrifuge tube, two the pipe.2000 leave the heart 10 min, shift supernatant again to the 1.5mL centrifuge tube, keep supernatant;

(2) with containing 2 mLPBS washed cells, according to per 10 ⁵The amount of cell/2 μ L adds DC surface markers CD80, CD86, and CD83, HLA-DR, 4 ℃ of following lucifuges are hatched 30 min;

(3) discard dyestuff, once with 2 mL PBS washed cells;

(4) with 2 mL PBS re-suspended cells, the paraformaldehyde that adds 1mL 2% is again fixed, and flow cytometer showed is subsequent use.

(5) detect with flow cytometer.The FACS data are used the CELLQuest software analysis.

1.5 FITC-dextran absorbs detection

The differentiation immature cell of collecting the 5th day is by 10 ⁵Cells/ml is suspended from the RPMI1640 complete medium, adds FITC-dextran (0.1 mg/mL, fluorescently-labeled glucosan) again; 37 ℃ of incubators are hatched 30 min, centrifugal 10 min of 1000 r/min, collecting cell; Clean 2 times with PBS, be resuspended in the streaming pipe at last, detect the fluorescence intensity of cell respectively; With 4 ℃ of conditions is benchmark, judges the antigen uptake ability of cell.

Conclusion: transverse axis is the fluorescence intensity behind the fluorescently-labeled glucosan of cytophagy (antigen) among Fig. 2, and it is strong more to engulf many more fluorescence intensities, and peak value more on the right side.The 37 ° of positive contrasts of C peak value in right side, the 4 ℃ of negative contrasts of peak value in left side.The result shows that the concentration along with chrysin increases, and peak value moves to left, and explains that fluorescence intensity weakens, and promptly engulfs antigenic capacity and weakens.Chrysin Chrysin suppresses the antigen uptake ability (Fig. 2) of immaturity dendritic cell; Can not effectively discern with antigen and can reduce to antigenic immune activation effect.Explain that it can suppress immune system to antigenic respond, in autoimmune disease, can reduce the ability of autoimmune cell identification autoantigen.Therefore can treat autoimmune disease;

1.6 Chrysin suppresses the allosome T cell proliferation (the cell-mediated relevant disease of treatment T) that ripe DC causes

Utilize the positive magnetic bead sorting allosome of CD4 CD4 positive cell, PBS washes twice, 37 ℃ of labelling CFSE (hydroxyl fluorescein diacetate butanimide fat) dyestuff (final concentration 2 μ M), 10 min, 2 * 10 ⁵Cell/200 μ l plants in round bottom 96 orifice plates; The ripe DC that handled by 1: 10,1: 20,1: 40 (DC/CD4+) adding ametycin (25 μ g/ml) and Chrysin again; After the co-cultivation 4 days; Collecting cell is done flow cytometer showed, with the decay number of times showed cell propagation variation of CFSE fluorescence intensity.

Conclusion: in immune system response, dendritic cell break up and increment to different effector T cells according to deriving from different antigenic signal stimulus T cells, and then mediate different immunoreation.Fig. 6 transverse axis be BMDC and T mixing with cells than (DC:T), the longitudinal axis is a T cell value added index.Chrysin (Chrysin) suppresses the allosome T cell proliferative response (CFSE method) that dendritic cell stimulate, and the T cell number reduces the relevant disease of meeting suppressor T cell mediation; It can treat the cell-mediated autoimmune diseasees of T such as multiple sclerosis;

1.7 the construction method of EAE mouse model

(1) laboratory animal and source

30 of female wild type C57BL/6 pathogen-free domestic (SPF level) mices, age in 6-8 week, body weight 18-20g; Available from Beijing Vital River Experimental Animals Technology Co., Ltd.; Raise the Experimental Animal Center in Medical University Of Tianjin, the feeding environment of mice is room temperature 20-25 ℃, and relative humidity is 40%-60%.

(2) preparation of antigen adjuvant emulsion

Mycobacterium (the Mycobacterium tuberculosis of 100 μ gMOG35-55 polypeptide and 500 μ g deactivations; Tubercule bacillus; Available from Difco company) mix emulsifying fully with 100 μ l normal saline and 100 μ l Freund ' s adjuvants (available from SIGMA company).

(3) experiment is divided into groups

Be the drug effect of research chrysin in the EAE in mice animal model, mice is divided four groups at random, and every group of 8 mices are respectively two normal saline matched groups and two 75 mg/kg/ days chrysin administration groups.A matched group and a chrysin administration group inspection record every day EAE in mice incidence and clinical function of nervous system are classified to behind the immune induction 35 days; Spinal cord and spleen are got in another matched group and chrysin administration group execution in the 15th day after the EAE in mice onset peak period is immune induction.

(4) experimental technique and step

A. 2 points of the subcutaneous branch of mice back are injected emulsifying agent (100 μ l/ point).

B. the while is at the pertussis toxin, PT of tail vein injection 200ng on the same day.

C. immunity every mice tail vein injection 200ng pertussis toxin, PT (pertussis toxin is available from List Biological Laboratories) once more after 48 hours.

D. to use chrysin gastric infusion, dosage every day be 100 mg/kg/ days to the beginning in the 7th day behind the immune induction of chrysin administration group.Mice begins to take place EAE after e 7-14 days.Every day inspection record EAE in mice incidence and the classification of clinical function of nervous system.

(5) EAE mouse model clinical score standard

Mice after the MOG immunity, the next day adopt blind method by two observers, adopt 5 fens international marking systems that mice is carried out clinical score, the clinical function of nervous system classification of every day inspection and record disease was until back 35 days of MOG immunity.The EAE in mice animal model function of nervous system concrete standard of marking is following: 0 minute, Non Apparent Abnormality did not have tangible clinical symptoms; 0.5 divide, the part tail is unable lax; 1 minute, the tail paralysis was visible slight clumsy fully; 2 minutes, back myasthenia of limbs was slow in action, mild ataxia; 2.5 divide acroparalysis after one; 3 minutes, acroparalysis behind the bilateral can not be recovered after passive the standing up, but can move after stimulating; 3.5 divide acroparalysis behind the bilateral, preceding myasthenia of limbs; 4 minutes, acroparalysis before the acroparalysis companion behind the bilateral; 5 minutes, moribund condition or death.

1.8 EAE mouse model onset peak period is got myeloid tissue and is done paraffin section dyeing

(1) spinal cord is fixed: behind immune induction, got myeloid tissue in the 15 day, tissue is placed embedded box, 4% neutral paraformaldehyde is fixed, and 30 min are above or spend the night.

(2) tissue dewatering: in the flowing water flushing myeloid tissue formaldehyde 30min → 75% ethanol 30min → 85% ethanol 30min → 95% ethanol I 1h → 95% ethanol II 2h → 95% ethanol III is spent the night or 2h → anhydrous alcohol I 30min → anhydrous alcohol II 30min → anhydrous alcohol III 30min

(3) transparency of organization (low temperature): xylene I 15 min → xylene II 10 min → xylene III 10 min

(4) tissue waxdip (60 ℃): stone (melting) wax I 90 min → stone (melting) wax II 60 min → open embedded box; Take out tissue; Put into the mould (comprising molten wax) of preheating, embedded box is put top → continuation and is added stone (melting) wax, until the embedded box lower submerged in stone (melting) wax.

(5) cooling: take out mould, place 4 ℃ of refrigerator overnight, fully after the cooling, room temperature storage is subsequent use

(6) paraffin section: spinal cord slice thickness is 10um, and wax disk(-sc) is put into 45 ℃ of warm water, and it is launched naturally.Hold the anticreep slide and fish for wax disk(-sc), then 60 ℃ of baking sheet >=4h.

(7) myeloid tissue's paraffin section HE dyeing

A.1 paraffin section de-waxing: paraffin section → xylene I 10 min → xylene II 10 min → xylene III 10 min

B. paraffin section aquation: 100% ethanol 2min → 95% ethanol 2min → 85% ethanol 2min → 75% ethanol 2min → distilled water flushing 1min

C. paraffin section HE dyeing: hematoxylin 10 min → tap water rinsing 1～3s → hydrochloride alcohol breaks up rapidly; (1 concentrated hydrochloric acid adds among the 70% ethanol 100ml) 1～3s → 0.1% ammonia/PBS; (7.4～8.0) are returned blue 30s～1min → tap water flushing 2-5min → distilled water rinsing 1～3s → 0.5% Yihong dyeing 1min → distilled water rinsing 1～3s → 75% ethanol, 10～30s → 85% ethanol, 10～30s → 95% ethanol 30s～1min → anhydrous alcohol 2～3min → transparent envelope and are hidden: xylene I 3-5min → xylene II 3-5 min →; (xylene is moistening) dripped natural gum → add coverslip.

(8) paraffin section Luxol fast blue dyeing: paraffin section de-waxing → dehydration to 95% ethanol → place Luxol to consolidate blue solution; Under 60 ℃ of temperature; Incubated overnight (16-24 h) → taking-up section is embathed in 95% alcoholic solution, the too much dye liquor of flush away → taking-up section; In deionized water, embathe → cut into slices and in 0.05% lithium carbonate solution, embathe 3-10s → taking-up section immediately fast; Place 70% alcoholic solution to break up, can clearly distinguish → take out section, in deionized water, wash → cut into slices and in 0.05% lithium carbonate solution, embathe gently for several times up to grey matter and white matter; Place 70% alcoholic solution to embathe for several times then; Become aeruginous up to the white matter color transition, during several no color of grey matter, stop in the differentiation → section deionized water flushing complete 95% ethanol 30s～1min → anhydrous alcohol 2～3min → transparent envelope and hide: xylene I 3-5min → xylene II 3-5 min → (xylene is moistening) dripped natural gum → add coverslip.

Result: DC can induce the T cells activation, is the moving person of beginning of body specific immune response.The effect of DC in autoimmune disease takes place also more and more paid attention to by people.Experiment shows that chrysin can suppress the differentiation (Fig. 1,2) of people's immunity BMDC; The antigen uptake ability (Fig. 3) that can suppress the immaturity dendritic cell; Suppress the Maturity (Fig. 4,5) of the activated people's immunity of LPS BMDC, the angtigen presentation ability that suppresses mature dendritic cell promptly suppresses the T cell proliferative response (Fig. 6) that dendritic cell stimulate.Experimental autoimmune encephalomyelitis is the disease animal model of marrowbrain inflammation property diseases such as people's multiple sclerosis, optic neuromyelitis and acute disseminated encephalomyelitis, also is the typical animal model of research LADA and diseases associated with inflammation.Every day of the present invention, the chrysin of per kilogram of body weight feeding 75mg can significantly suppress the occurring degree (Fig. 7) of mouse experiment systemic autoimmune encephalomyelitis; The best result and the clinical score summation (Fig. 8,9) that suppress the morbidity of mouse experiment systemic autoimmune encephalomyelitis; Alleviated the infiltration of inflammatory cell in the mouse experiment systemic autoimmune encephalomyelitis myeloid tissue and the degree (Figure 10,11) of demyelination, mouse experiment systemic autoimmune encephalomyelitis has been had the obvious treatment effect.Comprehensive The above results explains that chrysin can suppress immune system activation, can treat nervous system inflammation property diseases such as autoimmune inflammation property disease, particularly multiple sclerosis, optic neuromyelitis and acute disseminated encephalomyelitis.

Embodiment 2

Chrysin 50g and 280g starch mix homogeneously with starch slurry (get starch 220g water and process starch slurry) system granule, sieve, and drying is encapsulated.

Embodiment 3

Chrysin 80g and starch 340g mix homogeneously with starch slurry (get starch 210g water and process starch slurry) system granule, sieve, and drying adds 6 ‰ magnesium stearate, mixing, and compacting is wrapped film-coat and is promptly got in flakes.

Embodiment 4

Chrysin 100g, starch 400g and cellulose sieve in right amount; And fully mix, an amount of polyvinylpyrrolidonesolution solution is mixed with above-mentioned powder, sieve; Make wet granular in 60 ℃ of dryings; With the carboxymethyl starch sodium salt, 6 ‰ magnesium stearate and Pulvis Talci sieve in advance, join tabletting in the above-mentioned granule then.

Claims (6)

1. the application of chrysin aspect preparation conduct treatment autoimmune disease, nervous system inflammation property disease medicament.

2. chrysin suppresses the application in the immunologic rejection medicine in preparation as the treatment organ transplantation.

3. the described application of claim 1, wherein said autoimmune disease refers to: the autoimmune disease that immune BMDC or T cell are relevant.

4. the described application of claim 3, the autoimmune disease that wherein immune BMDC or T cell are relevant refers to: multiple sclerosis, rheumatoid arthritis, LADA retinitis, optic neuromyelitis, systemic lupus erythematosus (sle), struma lymphomatosa, autoimmune hemolytic anemia, AT, myasthenia gravis, primary biliary cirrhosis, aggressive chronic hepatitis, chronic glomerulus inflammation, myositis, systemic sclerosis.

5. the described application of claim 1, wherein nervous system inflammation property disease refers to multiple sclerosis, optic neuromyelitis and acute disseminated encephalomyelitis.

6. the described application of claim 5, its chrysin effective dose according to the 50-60Kg body weight is: 3.75g-4.5g/ days.

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