CN102670585A - Application of bavachinin for resisting asthma - Google Patents
Application of bavachinin for resisting asthma Download PDFInfo
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- CN102670585A CN102670585A CN2011100657039A CN201110065703A CN102670585A CN 102670585 A CN102670585 A CN 102670585A CN 2011100657039 A CN2011100657039 A CN 2011100657039A CN 201110065703 A CN201110065703 A CN 201110065703A CN 102670585 A CN102670585 A CN 102670585A
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Abstract
The invention discloses an anti-asthmatic drug, and belongs to the field of research and development of new drugs. The immune balance of the body is regulated by an immune system through different cytokines such as TNF-alpha (tumor necrosis factor-alpha), IL-4 (interleukin-4), INF-gamma (interferon-gamma). By establishing an IL-4-GFP (green fluorescence protein) transgene, a high-throughput screening model and an asthma model of mice are reported, and small-molecule drugs which can inhibit the secretion of IL-4 are screened. A small molecule compound which can inhibit the secretion of IL-4 and has an anti-asthmatic effect is found and named as # 29, and is shown in a structural formula (I). After the combination of IL-4 and a receptor IL-4R of the IL-4, downstream signals are collected for transduction to activate effectors and activate the proliferation and differentiation of B cells and eosinophils. The production of excess IL-4 can be mediated and accelerate various diseases, so that the reduction in the content of the IL-4 is an effective treatment means of inflammation, infection, immune or malignant diseases such as asthma and psoriasis. The small-molecule drug can inhibit the expression of the IL-4, and thus is expected to be developed into the anti-asthmatic drug.
Description
Technical field
The present invention relates to the application of bavachinin on the asthma medicine, the particularly development and application of the chemicals relevant with immune disease.
Background technology
The new drug development relevant with autoimmune disease has broad prospects.Wherein, China resident's health in immune disease serious threats such as inflammation diseases such as systemic lupus erythematosus (sle), rheumatic arthritis, asthma, enteritis, atherosclerosis, cancer and neurodegenerative diseases parkinson.Disease be that human immune system's adjusting is unbalance.Various immunologic processes are regulated and excited to immune system mainly through some cytokines of secretion (like IL-4, IL-6, IL-10, IL-12, TGF-β, NF-κ B, TNF-α etc.).Initial CD4+T cell is induced down in the different cells factor, is divided into different responsiveness helper T lymphocyte (Th) of function and phenotype and regulatory T cells (Treg), and its atomization receives the adjusting of cytokine.The Th2 cell all can produce IL-4 under the stimulation of ConA, IL-2, IL-4; IL-4 is with after its receptor IL-4R combines; Can induce different biological effects, raise signal transduction, the activation effect factor in downstream, activate B cell and eosinophilic granulocyte's propagation and differentiation.The generation of excessive IL-4 can mediate and quicken multiple disease, and therefore reducing IL-4 content or suppressing the NF-kB activity is effective treatment means of some inflammation, infection, immunity or malignant disease, for example asthma, psoriasis, inflammatory bowel or the like in theory.Therefore, we are based on the regulatory mechanism of IL-4, and the activation and the secretion of the artificial various immune cell factors of intervention are raised or downward modulation reaches proper proportion, thereby regulate and control some immune imbalance, reach the purpose of cure diseases.We have set up IL-4-GFP transgene report mice high flux screening model, and screening can suppress the excretory high activity small-molecule drug of IL-4.
Summary of the invention
The invention discloses the small-molecule drug 29# that can suppress the IL-4 secretion, have the asthma effect, relate to the relevant chemicals of autoimmune diseasees such as asthma, psoriasis and the application of anti-inflammatory drug.Its chemical structural formula of 29# and physical constant are seen table 1:
Table 1
The present invention is at the external IL-4-GFP transgene report mice high flux screening model that passes through; Adopt the full spleen cell of IL-4-GFP transgene report mice; Under the stimulation of ConA, IL-2, the IL-4 factor, to cultivate altogether 48 hours with the 29# of the variable concentrations of gradient dilution, cell is received in the back; With anti-CD4-APC cultured cells is dyeed respectively, carry out flow cytometer showed 29# at last IL-4-GFP
+The influence of amount.The present invention has not only proved that at cellular level 29# has significant inhibition effect to the generation of IL-4, through making up mouse asthmatic model, has confirmed in vivo that the 29# chemical compound has good asthma antiinflammatory action simultaneously.
The specific embodiment
Embodiment one: the activity of anti-asthmatic medicament 29# and toxicity assessment
1. external activity screening
1.1 with 29# gradient dilution successively in containing the modified form RPMI-1640 culture fluid of 10% hyclone, respectively get 100 μ l and add in 96 orifice plates, it is matched group that 0.02%DMSO is set.Getting changes IL-4-GFP gene report mouse spleen, in super-clean bench, grinds and obtains the lymphocyte suspension, and using the modified form RPMI-1640 culture fluid that contains 10% hyclone to process density is 4 * 10
6The cell suspension of individual cell/ml adds ConA, IL-2, the IL-4 factor differentiation that stimulates cellular proliferation, and the parallel adding of cell has been added in 96 orifice plates of variable concentrations small-molecule drug, and every hole 100 μ l cell suspension were cultivated 48 hours.
1.2 after 48 hours, receive and cultivate cultured cells in the streaming pipe, wash one time and (add PBS; 4 ℃, 1400 rev/mins, centrifugal 7 minutes); Dyeed 15 minutes with anti-CD4APC antibody ice bath after abandoning supernatant, and then wash one time, carry out flow cytometer at last and detect with PBS.
1.3 detecting datagram, streaming sees Fig. 1.
1.4 the docs-effect result sees Fig. 2.
2. toxotest (MTT)
2.1 earlier with 29# gradient dilution successively in containing the modified form RPMI-1640 culture fluid of 10% hyclone, respectively get 100 μ l and add in 96 orifice plates, it is matched group that 0.02%DMSO and water are set.Get wild type BALB/c mouse spleen, under super-clean bench, grind spleen and obtain spleen cell, using the modified form RPMI-1640 culture fluid that contains 10% hyclone to be mixed with density is 4 * 10
6The cell suspension of individual cells/ml adds ConA, IL-2, the IL-4 factor differentiation that stimulates cellular proliferation, and the cell suspension adding has been added in 96 orifice plates of variable concentrations small-molecule drug every hole 100 μ l.
2.2 after cultivating 48 hours, every hole is inhaled and is abandoned supernatant 120 μ l, adds the every hole 20 μ l of MTT solution, cultivates 4 hours again.Take out back 4 ℃, 1400r/min, centrifugal 7min, all supernatants of sucking-off, and every hole adds 100 μ l DMSO were put on the shaking table vibration (200r/min) 30 minutes, and purple crystal all dissolves.Measure the light absorption value in each hole at enzyme-linked immunosorbent assay instrument 570nm place.
2.3 the dosage of 29#---toxicity result sees Fig. 3.
3. activity in vivo evaluation
3.1 use wild-type mice; Carry out the pure albumen of the emulsive chicken of lumbar injection aluminium hydroxide (OVA) and make up mouse asthmatic model; On making up successful animal model, carry out evaluating drug effect in the body then: matched group lumbar injection PBS; The medication group is injected the 29# medicinal liquid of 100mg/kg body weight, 50mg/kg body weight respectively, lung tissue pathology section result such as Fig. 4.
3.2 the variation of real-time quantitative PCR analysis of control group and medication group lung tissue IL-4mRNA, result such as Fig. 5.
Description of drawings
1, the streaming result shows, the 29# chemical compound can the fine secretion (seeing Figure of description file map 1) that suppresses IL-4GFP in 5nM, 50nM concentration.
2, the docs-effect result shows, 29# suppresses obvious results to IL-4 when 5nM to 50nM concentration, have antiinflammatory, antasthmatic activity (seeing Figure of description file map 2).
3, the toxotest result shows, 29# has certain toxicity when the above concentration of 50nM, but less than the concentration of 10nM the time, does not have toxicity (seeing Figure of description file map 3).
4, through the pathological section analysis, can clearly observe the 29# chemical compound has good therapeutical effect to asthma, and at 100mg/kg dosage antiinflammatory action than 50mg/kg dosage effect obvious (seeing Figure of description file map 4).
5, the real-time quantitative PCR result shows that the 29# chemical compound has the trend that suppresses IL-4 generation in the asthmatic model mice body, has antasthmatic effect (seeing Figure of description file map 5).
Claims (6)
1. the invention discloses bavachinin (29#) and can suppress the IL-4 secretion, have the asthma effect, relate to the relevant chemicals of autoimmune diseasees such as asthma, psoriasis and the application of anti-inflammatory drug.Its chemical structural formula of 29# and physical constant are seen table 1:
Table 1
The present invention is at the external IL-4-GFP transgene report mice high flux screening model that passes through; Adopt the full spleen cell of IL-4-GFP transgene report mice; Under the stimulation of ConA, IL-2, the IL-4 factor, to cultivate altogether 48 hours with the 29# of the variable concentrations of gradient dilution, cell is received in the back; With anti-CD4-APC cultured cells is dyeed respectively, carry out flow cytometer showed 29# at last IL-4-GFP
+The influence of amount.The present invention has not only proved that at cellular level 29# has significant inhibition effect to the generation of IL-4, through making up mouse asthmatic model, has confirmed in vivo that the 29# chemical compound has good asthma antiinflammatory action simultaneously.
2. cultivating system according to claim 1 is characterized in that it is to adopt 96 well culture plates, and 200 μ l systems are 37 ℃, CO in condition
2Concentration is 5%, cultivate in the moistening incubator.
3. according to claim 1ly cultivate altogether, it is characterized in that it is that to adopt the 29# that adds variable concentrations behind the 100 μ l gradient dilutions in every hole and the concentration of 100 μ l be 4*10
6Individual/ml cell suspension is cultivated altogether.
4. stimulating factor ConA according to claim 1, IL-2, IL-4 is characterized in that its final concentration is respectively 2.5 μ g/ml, 2ng/ml, 20ng/ml.
5. anti-CD4-APC antibody staining according to claim 1 is characterized in that it is ice bath lucifuge dyeing 15 to 30 minutes.
6. asthma model according to claim 1 is characterized in that it is to the mouse peritoneal injection aluminium hydroxide pure albumen of emulsifying chicken (OVA) and inductive asthmatic model.
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| CN2011100657039A CN102670585A (en) | 2011-03-18 | 2011-03-18 | Application of bavachinin for resisting asthma |
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| CN2011100657039A CN102670585A (en) | 2011-03-18 | 2011-03-18 | Application of bavachinin for resisting asthma |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146804A (en) * | 2013-02-04 | 2013-06-12 | 中国科学院大连化学物理研究所 | Specificity probe zymolyte of glucuronic acid transferase UGT1A1 and application |
| CN103724309A (en) * | 2013-12-23 | 2014-04-16 | 天津科技大学 | Fructus psoraleae dihydroflavone methyl ether hydroxylation derivative and synthesis method thereof |
| CN104109144A (en) * | 2013-04-16 | 2014-10-22 | 上海中医药大学 | PPAR alpha/gamma dual agonist and its application |
| CN111773209A (en) * | 2020-06-16 | 2020-10-16 | 浙江中医药大学 | Application of psoralen in preparation of medicine for preventing or treating cochlear hair cell injury |
-
2011
- 2011-03-18 CN CN2011100657039A patent/CN102670585A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146804A (en) * | 2013-02-04 | 2013-06-12 | 中国科学院大连化学物理研究所 | Specificity probe zymolyte of glucuronic acid transferase UGT1A1 and application |
| CN103146804B (en) * | 2013-02-04 | 2015-04-22 | 中国科学院大连化学物理研究所 | Specificity probe zymolyte of glucuronic acid transferase UGT1A1 and application |
| CN104109144A (en) * | 2013-04-16 | 2014-10-22 | 上海中医药大学 | PPAR alpha/gamma dual agonist and its application |
| WO2014169800A1 (en) * | 2013-04-16 | 2014-10-23 | Shanghai University Of Traditional Chinese Medicine | A PPAR α/γ DUAL AGONIST AND ITS APPLICATION |
| CN103724309A (en) * | 2013-12-23 | 2014-04-16 | 天津科技大学 | Fructus psoraleae dihydroflavone methyl ether hydroxylation derivative and synthesis method thereof |
| CN103724309B (en) * | 2013-12-23 | 2016-04-06 | 天津科技大学 | A kind of Corylifolin methyl ether hydroxylation derivative and synthetic method |
| CN111773209A (en) * | 2020-06-16 | 2020-10-16 | 浙江中医药大学 | Application of psoralen in preparation of medicine for preventing or treating cochlear hair cell injury |
| CN111773209B (en) * | 2020-06-16 | 2022-06-17 | 浙江中医药大学 | Application of psoralen in preparation of medicine for preventing or treating cochlear hair cell injury |
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Application publication date: 20120919 |