WO2021135654A1 - 双硫仑在制备防治nlrp3炎症小体相关疾病的药物中的应用 - Google Patents

双硫仑在制备防治nlrp3炎症小体相关疾病的药物中的应用 Download PDF

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WO2021135654A1
WO2021135654A1 PCT/CN2020/127775 CN2020127775W WO2021135654A1 WO 2021135654 A1 WO2021135654 A1 WO 2021135654A1 CN 2020127775 W CN2020127775 W CN 2020127775W WO 2021135654 A1 WO2021135654 A1 WO 2021135654A1
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nlrp3 inflammasome
disease
disulfiram
peritonitis
arthritis
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PCT/CN2020/127775
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French (fr)
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胡文辉
杨忠金
孙平
邓文敏
熊兮
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广州医科大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/145—Amines having sulfur, e.g. thiurams (>N—C(S)—S—C(S)—N< and >N—C(S)—S—S—C(S)—N<), Sulfinylamines (—N=SO), Sulfonylamines (—N=SO2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

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  • the invention relates to the field of medicine, in particular to the application of disulfiram in the preparation of drugs for preventing and treating NLRP3 inflammasome-related diseases.
  • NLRP3 inflammasome is a pattern recognition receptor (PRR), which is the inflammatory activation platform of Caspase-1. It can recognize exogenous pathogen-associated molecular patterns (PAMP) such as lipopolysaccharide LPS, viral RNA or endogenous damage-associated molecular patterns (DAMP) such as DNA and endotoxin , Uric acid, ATP, A ⁇ and cell debris.
  • PAMP pathogen-associated molecular patterns
  • DAMP endogenous damage-associated molecular patterns
  • the purpose of the present invention is to provide an application of disulfiram in the preparation of drugs for preventing and treating NLRP3 inflammasome-related diseases.
  • Another object of the present invention is to provide a medicine for preventing and treating NLRP3 inflammasome-related diseases, including disulfiram and pharmaceutically acceptable excipients.
  • the present invention has the following beneficial effects:
  • DSF disulfiram
  • the structural formula of disulfiram is Its molecular formula is C 10 H 20 N 2 S 4 , molecular weight is 297, and CAS number is 97-77-8.
  • Disulfiram is a traditional drug for abstinence from alcohol. It is safe, non-toxic and has strong pharmacological effects. Even if a small amount of alcohol is consumed after taking this drug, the body will have serious discomfort, and the purpose of alcohol withdrawal can be achieved.
  • the inventors of the present invention unexpectedly discovered in the research that disulfiram can effectively inhibit the activation of NLRP3 inflammasomes and inhibit the maturation and secretion of the inflammation activation signal molecule Caspase-1P20 and the inflammatory cytokine IL-1 ⁇ .
  • NLRP3 inflammasomes Related diseases have good prevention and treatment effects, especially for peritonitis and gouty arthritis, with significant prevention and treatment effects.
  • Figure 1 shows the effect of DSF on inhibiting the secretion of IL-1 ⁇ induced by Nigericin (A) and ATP (B) in a concentration-dependent manner;
  • FIG. 2 is a diagram showing the effect of immunoblotting (WB) detecting that DSF can inhibit the activation of Caspase-1 and the secretion of IL-1 ⁇ induced by Nigericin and ATP in a concentration-dependent manner;
  • WB immunoblotting
  • Figure 3 is a diagram showing the effect of DSF on inhibiting the production of IL-1 ⁇ in peritoneal fluid and serum in LPS-induced peritonitis;
  • Figure 4 is a graph showing the effect of DSF on MSU-induced gout.
  • This embodiment provides an application of disulfiram in the preparation of drugs for preventing and treating NLRP3 inflammasome-related diseases.
  • the NLRP3 inflammasome-related disease is an acute NLRP3 inflammasome-related disease or a chronic NLRP3 inflammasome-related disease.
  • the NLRP3 inflammasome-related disease is arthritis.
  • the arthritis is gouty arthritis or rheumatoid arthritis.
  • the NLRP3 inflammasome-related disease is pelvic inflammatory disease.
  • the pelvic inflammatory disease is acute pelvic inflammatory disease.
  • the pelvic inflammatory disease is peritonitis.
  • the peritonitis is acute peritonitis.
  • the NLRP3 inflammasome-related disease is enteritis, hepatitis, Behçet’s disease, familial cold spontaneous inflammatory syndrome, M ⁇ ckle-Wells syndrome, chronic infantile neurocutaneous and joint syndrome, neonatal seizures Multi-system inflammatory disease, contact hypersensitivity or sunburn induced by ultraviolet rays.
  • the NLRP3 inflammasome-related disease is silicosis, asbestos lung, silicosis, Parkinson's disease, depression, type II diabetes, non-alcoholic fatty liver disease, alcoholic liver disease, kidney disease, multiple sclerosis Disease, amyotrophic lateral sclerosis, asthma or acute respiratory distress syndrome.
  • This embodiment also provides a medicine for preventing and treating NLRP3 inflammasome-related diseases, including disulfiram and pharmaceutically acceptable excipients.
  • the dosage form of the drug is capsule, granule, injection, pill, syrup, powder, ointment, emulsion, solution, suspension or tincture.
  • the dosage form of the drug is an oral solution; and/or the administration mode of the drug is oral; and/or, the drug is used in mammals or humans.
  • the auxiliary materials include excipients, fillers, compatibilizers, binders, humectants, disintegrants, slow solvents, absorption accelerators, adsorbents, diluents, solubilizers, emulsifiers , Lubricant, wetting agent, suspending agent, flavoring agent or perfume.
  • auxiliary material may be selected from at least one of the following components:
  • Fillers or compatibilizers for example, starch, lactose, sucrose, glucose, mannitol and silicic acid;
  • Binders such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic;
  • Humectants for example, glycerin
  • Disintegrants for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate;
  • Soothing solvents such as paraffin wax
  • wetting agents such as cetyl alcohol and glyceryl monostearate
  • Lubricants for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof.
  • the dosage form may also contain buffering agents.
  • the liquid dosage form may contain an inert diluent, such as water or other solvents, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide And oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.
  • an inert diluent such as water or other solvents, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide And oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.
  • the composition may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.
  • the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • suspending agents for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • composition for parenteral injection may contain physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions may be included, such as water, ethanol, polyols, and suitable mixtures thereof.
  • test materials used in the following examples are all purchased from conventional biochemical reagent stores.
  • DSF (Disulfiram) was purchased from Aladdin Company.
  • LPS and ATP were purchased from Sigma Company.
  • Nigericin (Nigericin) was purchased from Invitrogen.
  • Anti-mouse IL-1 ⁇ antibody (AF-401-NA) was purchased from R&D Company.
  • Anti-NLRP3 antibody (AG-20B-0014), anti-ASC antibody (AG-25B-0006) and anti-Caspase-1 antibody (AG-20B-0042) were purchased from Adipogen.
  • Anti- ⁇ -actin antibody (P30002) was purchased from Abmart.
  • ELISA kit (IL-1 ⁇ ) was purchased from InvivoGen.
  • C57BL/6J female mice were purchased from Guangdong Experimental Animal Center.
  • J774A.1 cells were purchased from jennio-bio.
  • the inventors of the present invention first performed in vitro experiments with disulfiram to inhibit NLRP3 inflammasomes, and then established an animal model of acute NLRP3 inflammasome-related diseases, and then used disulfiram for prevention and Treatment to observe the anti-inflammatory effects of disulfiram at the animal level.
  • step 2 collect the cell supernatant and use the Mo ⁇ se IL-1 ⁇ ELISA kit to determine the IL-1 ⁇ content.
  • the operation steps are carried out in accordance with the kit instructions. The results are shown in Figure 1.
  • LPS bacterial lipopolysaccharide
  • step 2 collect the cell supernatant (Supernant) and cell lysate (Lysate), extract the protein from the supernatant and lysate according to conventional methods, and use anti-IL-1 ⁇ antibody, anti-Caspase-1 antibody, anti-NLRP3 antibody and anti- ASC antibody was subjected to western blot analysis, and the results are shown in Figure 2.
  • Figure 2 shows that under the combined action of LPS pretreatment and the second signal Nigericin, the inflammasome is activated, and P20 and IL-1 ⁇ mature and secrete into the supernatant. With the addition of different concentrations of DSF, it effectively inhibits P20. And IL-1 ⁇ matures and secretes, and this inhibitory effect is dose-dependent.
  • mice were killed by cervical dislocation.
  • the blood was placed at room temperature to stand for 30 minutes, and then centrifuged at 3000 rpm for 30 minutes to collect serum;
  • Model group joint injection of 0.2mL sodium urate crystals (MSU); model administration group 1: same as the model group's modeling procedure, but oral DSF (25mg/kg) 1 hour after modeling, continuous feeding for 3 days; model administration Drug group 2: The same procedure as the model group, but oral administration of DSF (100mg/kg) 1 hour after modeling, continuous feeding for 3 days.
  • MSU sodium urate crystals
  • DSF improved the degree of swelling of bare joints in rats, and effectively inhibited the expression of IL-1 ⁇ in serum and caspase-1 in synovial tissue.
  • Synovial edema and fibrous tissue proliferation in DSF-treated mice Focal necrosis, lymphocyte and macrophage infiltration, and local periosteum involvement were all reduced.
  • disulfiram can effectively inhibit the maturation and secretion of the inflammatory activation signal molecule Caspase-1P20 and the inflammatory cytokine IL-1 ⁇ formed by the activation of NLRP3 inflammasome, and has a good prevention and treatment for NLRP3 inflammasome-related diseases.
  • the effect especially for peritonitis and gouty arthritis, has a significant effect on prevention and treatment.

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Abstract

一种双硫仑在制备防治NLRP3炎症小体相关疾病的药物中的应用。在研究中发现，双硫仑可以有效地抑制NLRP3炎症小体活化，抑制炎症活化信号分子Caspase-1 P20和炎症细胞因子IL-1β的成熟和分泌，对于NLRP3炎症小体相关疾病具有良好的防治作用，尤其是对于腹膜炎和痛风性关节炎，具有显著的防治效果。

Description

双硫仑在制备防治NLRP3炎症小体相关疾病的药物中的应用 技术领域
本发明涉及医药领域，特别是涉及一种双硫仑在制备防治NLRP3炎症小体相关疾病的药物中的应用。
背景技术
NLRP3炎症小体是一种模式识别受体(pattern recognition receptor，PRR)，是Caspase-1的炎症性激活平台。它能够识别外源性的病原体相关分子模式(pathogen-associated molecμlar pattern，PAMP)如脂多糖LPS、病毒RNA或内源性的损伤相关分子模式(damage-associated molecμlar pattern，DAMP)如DNA、内毒素、尿酸、ATP、Aβ和细胞碎片等。这些物质异常的出现在细胞外或细胞内的某个部位，使组织的稳态受到刺激而激活NLRP3炎症小体，抵抗外源性或内源性因素对机体的损害，但过度激活的NLRP3炎症小体会导致多种疾病发生发展，例如痛风、2型糖尿病以及神经退行性疾病等。尽管在基础研究中许多抑制剂对NLRP3炎症小体相关的疾病都有一定的缓解作用，但目前临床上，仍缺乏抑制NLRP3炎症小体活化的药物。
发明内容
基于此，本发明的目的是提供一种双硫仑在制备防治NLRP3炎症小体相关疾病的药物中的应用。
具体技术方案如下：
双硫仑在制备防治NLRP3炎症小体相关疾病的药物中的应用。
本发明的另一目的是提供一种防治NLRP3炎症小体相关疾病的药物，包括双硫仑以及药学上可接受的辅料。
与现有技术相比，本发明具有以下有益效果：
双硫仑(disulfiram，DSF)的结构式为
Figure PCTCN2020127775-appb-000001
其分子式为C 10H 20N 2S 4，分子量为297，CAS号为97-77-8。双硫仑是一种传统的戒酒药物，安全无毒，药理作用强，服用该药后即使饮用少量的酒，身体也会产生严重不适，而达到戒酒的目的。
本发明的发明人在研究中意外地发现，双硫仑可以有效地抑制NLRP3炎症小体活化，抑制炎症活化信号分子Caspase-1P20和炎症细胞因子IL-1β的成熟和分泌，对于NLRP3炎症小体相关疾病具有良好的防治作用，尤其是对于腹膜炎和痛风性关节炎，具有显著的防治效果。
附图说明
图1为DSF可以浓度依赖性的抑制Nigericin(A)和ATP(B)诱导IL-1β的分泌的效果图；
图2为免疫印迹(WB)检测DSF可以浓度依赖性的抑制Nigericin和ATP诱导的Caspase-1的活化以及IL-1β的分泌的效果图；
图3为DSF抑制LPS诱导的腹膜炎中腹腔液和血清IL-1β的产生的效果图；
图4为DSF抑制MSU诱导的痛风的效果图。
具体实施方式
为了便于理解本发明，下面将参照实施例对本发明进行更全面的描述，以下给出了本发明的较佳实施例。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。本文所使用的术语“和 /或”包括一个或多个相关的所列项目的任意的和所有的组合。
本实施方式提供一种双硫仑在制备防治NLRP3炎症小体相关疾病的药物中的应用。
在其中一些实施例中，所述NLRP3炎症小体相关疾病为急性NLRP3炎症小体相关疾病或慢性NLRP3炎症小体相关疾病。
在其中一些实施例中，所述NLRP3炎症小体相关疾病为关节炎。
在其中一些实施例中，所述关节炎为痛风性关节炎或类风湿性关节炎。
在其中一些实施例中，所述NLRP3炎症小体相关疾病为盆腔炎。
在其中一些实施例中，所述盆腔炎为急性盆腔炎。
在其中一些实施例中，所述盆腔炎为腹膜炎。
在其中一些实施例中，所述腹膜炎为急性腹膜炎。
在其中一些实施例中，所述NLRP3炎症小体相关疾病为肠炎、肝炎、白塞病、家族性冷自发炎症综合征、Mμckle-Wells综合征、慢性婴儿神经皮肤和关节综合征、新生儿发作多系统炎性疾病、接触性超敏反应或紫外线所诱导的皮肤晒伤。
在其中一些实施例中，所述NLRP3炎症小体相关疾病为硅肺、石棉肺、矽肺、帕金森病、抑郁症、II型糖尿病、非酒精性脂肪肝病、酒精性肝病、肾病、多发性硬化症、肌萎缩侧索硬化、哮喘或急性呼吸窘迫综合征。
本实施方式还提供一种防治NLRP3炎症小体相关疾病的药物，包括双硫仑以及药学上可接受的辅料。
在其中一些实施例中，所述药物的剂型为胶囊剂、颗粒剂、注射剂、丸剂、糖浆剂、散剂、膏剂、乳液、溶液、悬浮液或酊剂。
在其中一些实施例中，所述药物的剂型为口服溶液；和/或，所述药物的给药方式为口服；和/或，所述药物用于哺乳动物或人。
在其中一些实施例中，所述辅料包括赋形剂、填料、增容剂、粘合剂、保湿剂、崩解剂、缓溶剂、吸收加速剂、吸附剂、稀释剂、增溶剂、乳化剂、润滑剂、润湿剂、悬浮剂、矫味剂或香料。
具体地，所述辅料可选自以下组分中的至少一种：
(a)填料或增容剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；
(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶；
(c)保湿剂，例如，甘油；
(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；
(e)缓溶剂，例如石蜡；
(f)吸收加速剂，例如，季胺化合物；
(g)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯；
(h)吸附剂，例如，高岭土；
(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。
除了活性成分外，液体剂型可包含惰性稀释剂，如水或其它溶剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。
除了活性成分外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。可包含适宜的含水和非水载体、稀释剂、溶剂或赋形剂，例如水、乙醇、多元醇及其适宜的混合物。
以下结合具体实施例对本发明作进一步详细的说明。
下述实施例中的实验方法，如无特殊说明，均为常规实验方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。
DSF(双硫仑)购自aladdin公司。
LPS和ATP购自Sigma公司。
Nigericin(尼日利亚菌素)购自Invitrogen公司。
抗鼠IL-1β抗体(AF-401-NA)购自R&D公司。
抗NLRP3抗体(AG-20B-0014)、抗ASC抗体(AG-25B-0006)和抗Caspase-1抗体(AG-20B-0042)购自Adipogen公司。
抗β-actin抗体(P30002)购自Abmart公司。
ELISA试剂盒(IL-1β)购自InvivoGen。
C57BL/6J雌性小鼠购自广东省实验动物中心。
J774A.1细胞购自jennio-bio。
为了验证双硫仑的效果，本发明的发明人首先在体外实验进行了双硫仑对NLRP3炎症小体抑制，然后建立了急性NLRP3炎症小体相关疾病动物模型，再用双硫仑进行预防和治疗，以观察双硫仑在动物水平的抗炎症小体的作用。
实施例1 双硫仑对NLRP3炎症小体抑制作用的体外研究
1、第一天，将J774A.1细胞分到96孔板上，每个孔5×10 5个细胞；
2、种板过夜，弃上清，每孔加入100μL含细菌脂多糖(LPS)(1μg/ml)的含10％血清的DMEM培养基，然后加入不同浓度的(0μM、2μM、5μM、10μM)DSF处理1小时，再加入尼日利亚菌素(Nigericin，10μM)或ATP(5mM)处理1小时；
3、步骤2处理后，收集细胞上清液，采用Moμse IL-1βELISA试剂盒测定IL-1β含量，其操作步骤按照试剂盒说明书进行，结果见图1。
图1结果显示，在LPS预处理和第二信号Nigericin或ATP的共同作用下，炎症小体活化，IL-1β成熟并分泌到上清之中。随着不同浓度的DSF加入，有效地抑制IL-1β成熟和分泌，并且，这种抑制效果是剂量依赖的。
实施例2 DSF抑制NLRP3炎症小体的活化
1、第一天，将J774A.1细胞分到6孔板上，每个孔2×10 6个细胞；
2、种板过夜，弃上清，每孔加入1mL含细菌脂多糖(LPS)(1μg/ml)的含10％血清 的DMEM培养基，然后弃上清，加入不同浓度的(0μM、2μM、5μM、10μM)DSF的opti-MEM培养基处理1小时，再加入尼日利亚菌素(Nigericin，10μM)处理1小时；
3、步骤2处理后，收集细胞上清(Supernant)和细胞裂解液(Lysate)，上清和裂解液按常规方法提取蛋白，用抗IL-1β抗体、抗Caspase-1抗体、抗NLRP3抗体和抗ASC抗体进行western blot分析，结果见图2。
图2结果显示，在LPS预处理和第二信号Nigericin的共同作用下，炎症小体活化，P20和IL-1β成熟并分泌到上清之中，随着不同浓度的DSF加入，有效地抑制P20和IL-1β成熟和分泌，并且，这种抑制效果是剂量依赖的。
实施例3 双硫仑对NLRP3炎症小体抑制作用的体内研究——DSF抑制LPS诱导的腹膜炎
1、取6-8周龄C57BL/6雌鼠，称重，并进行编号。然后灌胃DSF，100mg/kg每只鼠，对照组灌胃含有10％DMSO的PBS，每天一次，连续三天，第三天灌胃DSF 1h后，腹腔注射LPS，20mg/kg每只鼠；
2、6小时后，摘除眼球放血，并收集血液，放血完毕后颈椎脱臼处死小鼠。血液放于室温静置30min，然后3000rpm离心30min，收集血清；
3、用1ml生理盐水冲洗腹腔，吸出的生理盐水，3000rpm离心5分钟，收集上清液；
4、步骤2得到的血清和步骤3得到的腹腔灌洗上清液用ELISA的方法进行IL-1β的测定，结果见图3。
从图3的结果看，腹腔注射LPS后，IL-1β等炎性细胞因子水平有明显的升高，而DSF的加入有效的抑制这些炎性因子的水平，表明DSF可以有效地抑制LPS诱导的腹腔炎。
实施例4 双硫仑对NLRP3炎症小体抑制作用的体内研究——DSF抑制MSU诱导的痛风
1、取6-8周龄的大鼠分为3组(模型组、模型给药1组和模型给药2组)，每组6只大鼠。模型组：关节注射0.2mL的尿酸钠结晶(MSU)；模型给药1 组：同模型组造模程序一致，但造模后1h口服DSF(25mg/kg)，连续喂药3天；模型给药2组：同模型组造模程序一致，但造模后1h口服DSF(100mg/kg)，连续喂药3天。
2、72小时后，测量关节肿胀程度；眼眶取血，分离血清；取各个大鼠踝关节滑膜组织HE染色制备切片，并检测滑膜组织中的caspase-1、血清中IL-1β的表达变化。结果见图4。
从图4的结果看，DSF改善大鼠裸关节肿胀程度，有效抑制了血清中的IL-1β和滑膜组织中的caspase-1的表达，DSF处理组小鼠的滑膜水肿、纤维组织增生、灶性坏死、淋巴细胞及巨噬细胞浸润以及局部骨膜受累均有所降低。这些结果表明DSF有效的抑制MSU诱导的痛风。
由上述实验可知，双硫仑可以有效地抑制NLRP3炎症小体活化所形成的炎症活化信号分子Caspase-1P20和炎症细胞因子IL-1β的成熟和分泌，对于NLRP3炎症小体相关疾病具有良好的防治作用，尤其是对于腹膜炎和痛风性关节炎，具有显著的防治效果。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 双硫仑在制备防治NLRP3炎症小体相关疾病的药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述NLRP3炎症小体相关疾病为关节炎。
  3. 根据权利要求2所述的应用，其特征在于，所述关节炎为痛风性关节炎或类风湿性关节炎。
  4. 根据权利要求1所述的应用，其特征在于，所述NLRP3炎症小体相关疾病为盆腔炎。
  5. 根据权利要求4所述的应用，其特征在于，所述盆腔炎为腹膜炎。
  6. 根据权利要求5所述的应用，其特征在于，所述腹膜炎为急性腹膜炎。
  7. 根据权利要求1所述的应用，其特征在于，所述NLRP3炎症小体相关疾病为肠炎、肝炎、硅肺、石棉肺、矽肺、白塞病、紫外线所诱导的皮肤晒伤、接触性超敏反应、帕金森病、抑郁症、II型糖尿病、非酒精性脂肪肝病、酒精性肝病、肾病、家族性冷自发炎症综合征、Mμckle-Wells综合征、慢性婴儿神经皮肤和关节综合征、新生儿发作多系统炎性疾病、多发性硬化症、肌萎缩侧索硬化、哮喘或急性呼吸窘迫综合征。
  8. 一种防治NLRP3炎症小体相关疾病的药物，其特征在于，包括双硫仑以及药学上可接受的辅料。
  9. 根据权利要求8所述的药物，其特征在于，所述药物的剂型为胶囊剂、颗粒剂、注射剂、丸剂、糖浆剂、散剂、膏剂、乳液、溶液、悬浮液或酊剂。
  10. 根据权利要求9所述的药物，其特征在于，所述辅料包括赋形剂、填料、增容剂、粘合剂、保湿剂、崩解剂、缓溶剂、吸收加速剂、吸附剂、稀释剂、增溶剂、乳化剂、润滑剂、润湿剂、悬浮剂、矫味剂或香料。
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