Technical field:

The present invention relates to biomedicine field, relate in particular to artemisinin derivative, the application of particularly a kind of artemisinin derivative in the medicine of preparation treatment Crohn disease.

Background technology:

In the prior art, the medicine that is used for the treatment of Crohn disease has glucocorticoid, 5-aminosalicylic acid, immunosuppressant such as azathioprine etc., anti-tumor necrosis factor-alpha monoclonal antibodies etc.Present therapeutic goal is a relief of symptoms, and keeps alleviation.

5-aminosalicylic acid: recommend to be used for light moderate patient, can keep treatment, side effect is minimum relatively, but not good to the symptom control of most of patients.

Glucocorticoid: be used for the out of contior severe patient of 5-aminosalicylic acid, energy relief of symptoms, but the mucosa ulcer of can not healing, side effect is more relatively, does not have and keeps mitigation.

Immunosuppressant: keep treatment after being used for invalid or dependence of hormone and hormone induction and alleviating, curative effect is far superior to 5-aminosalicylic acid and glucocorticoid, the mucosa ulcer of healing, but side effect is many, is not recommended as a line medication.The most frequently used in the immunosuppressant is azathioprine, side effect incidence rate height, especially bone marrow depression, and most Chinese patients can not withstand long term exposure, does not reach therapeutic purposes.

Anti-tumor necrosis factor-alpha monoclonal antibodies: its curative effect height, keep mitigation, prolonged application is costly, and general family can't bear; And must share with immunosuppressant or glucocorticoid, easy occurrence condition pathogenic infection, China is the tuberculosis hotspot, clinical practice is restricted, 30% patient is arranged approximately to its no response.

Crohn disease is an all-digestive tract nonspecific inflammation disease, etiology unknown, present result of study point out its be because of gut barrier impaired, certain micro-organisms enters intestinal mucosa, by dendritic cell, antigen presenting cell and macrophage picked-up, stimulate IL-12, IL-23, cytokine secretions such as IL-4, IL-6.Wherein IL-12 impels T cells to the Th1 cell differentiation, and justacrine gamma interferon, tumor necrosis factor-alpha etc. trigger the reaction of Th1 type; And IL-23 at TGF-β, impel T cells to the Th17 cell differentiation when IL-6 exists, justacrine IL-17, IL-6, tumor necrosis factor-alpha etc.The Th1/Th17 immunne response causes inflammation, and the immunodeficiency of intestinal mucosa makes inflammation continue not heal, chronic development.Nuclear Factor-Kappa B path overactivity in the inflammatory reaction, 5-aminosalicylic acid, glucocorticoid, tumor necrosis factor-alpha monoclonal antibody all have the activation of direct or indirect inhibition nuclear Factor-Kappa B and/or disturb the immunne response effect and reach therapeutic purposes.

Arteannuin is to separate the sesquiterpene lactones chemical compound that obtains from traditional Chinese herbal medicine Hemerocallis citrina Baroni Artemisia, is widely used in the malaria treatment, and the drug-fast Plasmodium falciparum of traditional antimalarial is had significant curative effect.Artemisinin derivatives such as dihydroarteannuin, Artemether, artesunate, arteether are effective equally to malaria.The instant effect of bringing down a fever of the fever that artemisinin-based drug causes for malaria, about 32h can bring down a fever, and other drug needs 2-3d, antagonism chloroquine malaria is also effective.In addition, high dose uses arteannuin that animal is had neurotoxicity and fetal toxicosis, but is applied to human comparatively safe.Studies show that in recent years, artemisinin-based drug has killing action to tumor cell, mainly realizes by apoptosis-induced approach, for normal cell no cytotoxicity almost.

The structural formula of artesunate is:

The structural formula of dihydroartemisinine is:

The structural formula of Artemether is:

The structural formula of SM905 is:

Summary of the invention:

The object of the present invention is to provide the application of a kind of artemisinin derivative in the medicine of preparation treatment Crohn disease, described this arteannuin and artemisinin derivative will solve big, the unfavorable technical problem of effect of prior art Chinese medicine treatment Crohn disease side effect.

The invention provides the application of a kind of artemisinin derivative in the medicine of preparation treatment Crohn disease.

Further, described artemisinin derivative is selected from artesunate or dihydroartemisinine or Artemether or SM905.

Artemisinin derivatives such as artesunate, dihydroartemisinine, Artemether, SM905 all have the activatory antiinflammatory action of the nuclear Factor-Kappa B of inhibition, simultaneously also can suppress the Th1/Th17 immunne response, demonstrate effectiveness in the clinical practice of treatment rheumatoid arthritis and systemic lupus erythematosus (sle), its immunosuppressive action can be used for treating Crohn disease.

The present invention and prior art contrast, and its effect is actively with tangible.Artemisinin derivative is by suppressing the Th1/Th17 immunne response, suppressing the purpose that the nuclear Factor-Kappa B activation reaches the treatment Crohn disease.Than existing medicine, the few side effects of artemisinin derivative and light, rarely seen indivedual case one property crossed reticulocytes descend and liver function injury in the treatment rheumatoid arthritis was followed up a case by regular visits in 1 year, and liver protecting therapy need not drug withdrawal and can recover.Therefore, artemisinin derivative can be taken for a long time for the patient, can keep treatment and reaches effective therapeutic purposes.

Description of drawings:

Fig. 1 is the photo of mice colon light microscopy checking in the ethanol matched group (amplifying 40 times).

Fig. 2 is the photo of mice colon light microscopy checking in the trinitro-benzene-sulfonic acid model group (amplifying 40 times).

Fig. 3 is the photo of mice colon light microscopy checking in the artesunate treatment group (amplifying 40 times).

Fig. 4 is the photo of mice colonic mucosa nuclear Factor-Kappa B p65 immunohistochemical staining (amplifying 400 times) in the ethanol matched group.

Fig. 5 is the photo of mice colonic mucosa nuclear Factor-Kappa B p65 immunohistochemical staining (amplifying 400 times) in the trinitro-benzene-sulfonic acid model group.

Fig. 6 is the photo of mice colonic mucosa nuclear Factor-Kappa B p65 immunohistochemical staining (amplifying 400 times) in the artesunate treatment group.

Fig. 7 is that biochemical process detects the active sketch map of three groups of mice colon myeloperoxidase (MPOies (MPO).

Fig. 8 adopts the ELISA test kit to detect three groups of mice colonic mucosa levels of tnf-alpha sketch maps

Fig. 9 adopts the ELISA test kit to detect three groups of mice colonic mucosa IL-17 content sketch maps

Figure 10 adopts the ELISA test kit to detect three groups of mice colonic mucosa gamma interferon content sketch maps.

The specific embodiment:

Embodiment 1: the foundation of animal grouping and colitis model

C57BL/6 mice (by zoopery center, Chinese Academy of Sciences Shanghai) is divided into 3 groups at random, is respectively ethanol matched group, trinitro-benzene-sulfonic acid model group and artesunate treatment group, 6 every group.Trinitro-benzene-sulfonic acid group and artesunate treatment group mouse colitis model are set up: after the slight anesthesia of mice, flexible pipe inserts the about 4cm of anus, slowly injects trinitro-benzene-sulfonic acid/alcoholic solution 0.1ml; The ethanol matched group injects alcoholic solution 0.1ml, is inverted about 60s, puts back in the cage.After the coloclysis about 8 hours, artesunate treatment group mouse peritoneal injection artesunate 150mg/kg/ days (south, Guilin medicine limited company), ethanol matched group and trinitro-benzene-sulfonic acid model group injection equal-volume sodium bicarbonate solution.Continuous 7 days, observe and record disease activity index (seeing Table 1).

Table 1:

Embodiment 2: histological examination

Test and put to death mice on the 8th day, get serum and be stored in-70 ℃ of refrigerators; Cut open the belly, get intestinal and be stored in the liquid nitrogen.The perusal colon changes and marks substantially, and standard is: colonic ulcer, colon and peritoneal adhesion, colon wall thickens, four parameters of mucosa edema, each parameter order of severity is divided 4 grades from normal (0 minute) to the most serious (4 minutes), and scoring is the meansigma methods of four parameter scores substantially.Get the most obvious colon of pathological changes and be fixed in 10% formalin solution, the routine paraffin wax embedding, section, haematoxylin-Yihong dyeing, om observation Histological change and scoring, standard is: 0 minute: NIP; 1 minute: the colon light inflammation; 2 minutes: a small amount of inflammatory cell such as infiltrations such as neutrophilic granulocyte, lymphocyte are arranged in the colon; 3 minutes: a large amount of inflammatory cells such as infiltrations such as neutrophilic granulocyte, lymphocyte, high vessel density, colon wall thickens; 4 minutes: the saturating wall of inflammatory cell soaked into, the goblet cell disappearance, and high vessel density, colon wall thicken (shown in Fig. 1, Fig. 2 and Fig. 3 and table 1).

Embodiment 3 immunohistochemical stainings and result judge

Adopt the SP method to carry out the nuclear factor-K B P 65 immunohistochemical staining, the concrete operations step is undertaken by nuclear factor-K B P 65 SP immunohistochemical staining test kit description (Fujian steps neoplasm technological development company limited).Result's judgement comprises two aspects: (1) positive staining strength grading: 0 grade negative, acellular dyeing; 1 grade is the weak positive, occurs the yellow particle that is dispersed in the cell; 2 grades is moderate positive, occurs a small amount of brown yellow granule in the cell; 3 grades: occur a large amount of brown yellow granules in the cell.(2) positive cell number classification: 0 grade negative, acellular dyeing; 1 grade of positive cell number≤10%; 2 grades of positive cell number 11%～50%; 3 grades of positive cell number 51%～80%; 4 grades of positive cell number＞80%.Positive mark's mark=positive staining strength grading * positive cell number classification.(shown in Fig. 4, Fig. 5 and Fig. 6 and Biao).

Table 1

Compare * p＜0.05 with artesunate treatment group; #p＜0.01

Mouse disease activity index, scoring substantially, histological score, nuclear Factor-Kappa B scoring have statistical significance in the comparison of artesunate treatment group and trinitro-benzene-sulfonic acid model group, but more also reach statistical significance with the ethanol matched group, illustrate that artesunate can effectively suppress mouse colitis disease, but fail to reach normal level.

Embodiment 4 ELISA detect colon's homogenate cell/inflammatory factor content

The homogenate on ice of a mice of clip colon, centrifuging and taking supernatant, Brandford method quantification of protein.Gamma interferon, IL-17, tumor necrosis factor-alpha are pressed the ELISA detection kit description (R﹠amp of gamma interferon, IL-17 and tumor necrosis factor-alpha respectively; The packing of D company) operation and drawing standard curve are tried to achieve each sample size, and the result represents with pg/g.

Mice colon myeloperoxidase (MPO) (MPO) activity, press MPO biochemical process detection kit description operation (as Fig. 7, Fig. 8, Fig. 9 and shown in Figure 10), MPO activity, gamma interferon, IL-17, the content of tumor necrosis factor-alpha in the mice colon compare p＜0.05 in artesunate treatment group and trinitro-benzene-sulfonic acid model group; Compare p＞0.05 with the ethanol matched group, the expression of MPO activity, gamma interferon, IL-17, tumor necrosis factor-alpha in the artesunate energy inflammation-inhibiting tissue is described, by suppressing the purpose that immunoreation reaches treatment mouse colitis disease.

The foregoing description all adopts SPSS11.5 statistics software kit, Kruskal Wallis check is adopted in the aggregate analysis of nonparametric data, the Wilcoxon rank test is adopted in relative analysis between group, and the ELISA testing result adopts one factor analysis of variance, and there is statistical significance p＜0.05 for difference; P＜0.01 is remarkable statistical significance for difference has.

The foregoing description has adopted artesunate to do experiment, and artesunate changes into dihydroartemisinine and plays a role in liver, so artesunate and dihydroartemisinine can both be treated Crohn disease.Artesunate or dihydroartemisinine tablet are oral, the 1-2 sheet, and a twice-daily～three time just can play excellent curative, and be suitable for long-term oral medication.The inventor adopts top same experimentation to test, find Artemether and SM905 also can the inflammation-inhibiting tissue in the expression of MPO activity, gamma interferon, IL-17, tumor necrosis factor-alpha, by suppressing the purpose that immunoreation reaches treatment mouse colitis disease, have the therapeutic effect same with artesunate.