CN109364058A - 富马酸酯及其可药用盐在制备治疗铁死亡相关疾病的药物中的应用 - Google Patents
富马酸酯及其可药用盐在制备治疗铁死亡相关疾病的药物中的应用 Download PDFInfo
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- CN109364058A CN109364058A CN201811480562.5A CN201811480562A CN109364058A CN 109364058 A CN109364058 A CN 109364058A CN 201811480562 A CN201811480562 A CN 201811480562A CN 109364058 A CN109364058 A CN 109364058A
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- fumarate
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- liver
- iron death
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Abstract
本发明涉及富马酸酯及其可药用盐在制备治疗铁死亡相关疾病的药物中的应用,所述富马酸酯包括富马酸二甲酯和/或富马酸单甲酯。本发明首次揭示了富马酸酯及其可药用盐对铁死亡关键调控基因GPX4蛋白水平的影响,为研究铁死亡疾病的治疗策略提供了理论依据,为制备新的铁死亡相关疾病的药物提供了一个嵌入点。
Description
技术领域
本发明属于化学医药领域,具体涉及富马酸酯及其可药用盐在制备治疗铁死亡相关疾病的药物中的应用。
背景技术
铁死亡是一类铁/活性氧簇依赖性的细胞死亡途径,不同于凋亡、坏死和自噬等其他死亡途径。铁死亡主要表现为细胞脂质过氧化和胞内活性氧簇累积。Ferrostatin-1和Erastin是目前公认的铁死亡抑制剂和激活剂。研究表明,Erastin通过抑制胱氨酸/谷氨酸转运体,阻碍胱氨酸被摄取还原为半胱氨酸,影响谷胱甘肽的合成。GSH是谷胱甘肽过氧化物酶发挥抑制脂质活性氧簇生成作用的必要辅助因子。其中谷胱甘肽过氧化酶4(GPX4)是重要的铁死亡调控因子,可以抑制细胞膜脂质过氧化的发生。GPX4的活性或表达受抑制时,可触发铁死亡,反之,其活性或表达增加可抑制铁死亡。
研究发现多种疾病与铁死亡相关,包括神经***疾病(帕金森病、脑室周围白质软化症、亨廷顿病、阿尔兹海默病、运动神经衰退和脑外伤等;肿瘤(头颈部恶性肿瘤、乳腺癌、食管癌、肝癌、胰腺癌、肾癌和弥漫性大B细胞淋巴瘤等);缺血再灌注损伤(心肌、肝和肾等);急性肾衰竭和铁代谢相关疾病(动脉粥样硬化和糖尿病等);肝脏疾病(血色沉着病、原发性胆汁性胆管炎、肝纤维化等)。因此,以铁死亡为靶点,通过可以抑制铁死亡的药物靶向抑制铁死亡,对铁死亡相关疾病的治疗具有重要意义。
富马酸二甲酯(dimethyl fumarate,DMF,商品名Tecfidera)是在2013年3月由美国食品药品监督管理局批准用于治疗多发硬化症的新型药物。DMF及其体内主要代谢活性产物富马酸单甲酯(monomethyl fumarate,MMF)可以通过激活在细胞抗氧化机制中发挥着重要作用的KEAP1-NRF2-ARE信号通路,抑制细胞ROS累积,从而对抗细胞过氧化损伤。
CN107253927A公开了一类磷脂氢谷胱甘肽过氧化物酶(GPX4)激活剂及应用,体外酶活测试和细胞模型实验证实,该化合物作为GPX4的激活剂能够用于治疗和预防各种炎症、氧化损伤、神经退行性病变及铁死亡相关疾病。
CN107890567A提供了CDO1的新应用,所述的新应用是CDO1应用于制备与铁死亡相关的胃癌治疗药物。CDO1在胃癌中是c-Myb信号通路的重要组成部分,是铁死亡过程的重要代谢节点。该发明首次揭示了CDO1调控半胱氨酸代谢在Erastin诱导的胃癌细胞铁死亡中的作用机制以及c-Myb在铁死亡过程中对CDO1表达的转录调控作用。过表达CDO1可促进胃癌细胞的铁死亡过程。因此,本发明为研究以铁死亡为基础的胃癌治疗提供了理论依据,为制备新的胃癌治疗药物提供了一个嵌入点。
CN108484527A公开了一种新的能够抑制铁死亡的10H-吩噻嗪类衍生物,通过对其结构优化和构效关系的研究,证实了该10H-吩噻嗪类衍生物能够对铁死亡产生较好的抑制作用,并且其中存在对大鼠局灶性脑缺血模型表现出较好的治疗效果的化合物,其能够作为制备铁死亡抑制剂的主要活性成分,该化合物和该化合物制备得到的抑制剂均具有很好的药用潜力;该发明提供的新化合物的制备方法简便,反应条件温和,便于操作和控制,能耗小,产率高,成本低,可适合产业化生产,制备得到的化合物生物活性较高,选择性强,类药性显著,具有广阔的市场前景。
现有技术公开的治疗铁死亡相关疾病的策略并不多,因此,开发出一种新的以铁死亡为靶点,选择性强,药效显著的治疗铁死亡相关疾病的药物是非常有意义的。
发明内容
针对现有技术存在的问题,本发明的目的在于提供富马酸酯及其可药用盐在制备治疗铁死亡相关疾病的药物中的应用。
为达到上述目的,本发明采用以下技术方案:
本发明提供了富马酸酯及其可药用盐在制备治疗铁死亡相关疾病的药物中的应用。
富马酸二甲酯及其体内主要代谢活性产物富马酸单甲酯可以通过激活在细胞抗氧化机制中发挥着重要作用的KEAP1-NRF2-ARE信号通路,抑制细胞ROS累积,从而对抗细胞过氧化损伤,而现有技术未公开过富马酸二甲酯及富马酸单甲酯在治疗铁死亡相关疾病中的应用,其作为一种以铁死亡为靶点的新型的治疗铁死亡相关疾病的药物具有广阔的应用前景。
在本发明中,所述富马酸酯包括富马酸二甲酯和/或富马酸单甲酯。
所述药物的剂型包括片剂、散剂、颗粒剂、胶囊剂、注射剂、喷雾剂、膜剂、栓剂、滴鼻剂或滴丸剂。
所述药物的给药途径包括静脉注射、腹腔注射、肌肉注射、皮下注射、口服给药、舌下给药、鼻腔给药或经皮给药。
在本发明中,所述铁死亡相关疾病包括肝脏疾病、神经***疾病、肿瘤、急性肾衰竭、缺血再灌注损伤或铁代谢相关疾病。
所述肝脏疾病是由铁死亡相关因子水平异常引起的肝脏疾病。
优选地,所述肝脏疾病包括血色沉着病、原发性胆汁性胆管炎、肝纤维化。
在本发明中,所述神经***疾病包括帕金森病、阿尔兹海默症、脑室周围白质软化症、亨廷顿病、运动神经衰退症或脑外伤。
所述肿瘤包括头颈部恶性肿瘤、乳腺癌、食管癌、肝癌、胰腺癌、肾癌或弥漫性大B细胞淋巴瘤。
所述缺血再灌注损伤包括心肌缺血再灌注损伤、肝缺血再灌注损伤或肾缺血再灌注损伤。
所述铁代谢相关疾病包括动脉粥样硬化或糖尿病。
在本发明中,所述富马酸酯及其可药用盐为负载于药用载体上的富马酸酯及其可药用盐。
富马酸酯及其可药用盐可以负载于常用药用载体上作为治疗铁死亡相关疾病的药物,实现更好的生物相容性、生物安全性和药效,所述常用药用载体包括脂质体、胶束、树枝状大分子、微球、微囊等等。
在本发明中,所述富马酸酯及其可药用盐为药物组合物中含有的富马酸酯及其可药用盐。
富马酸酯及其可药用盐也可以与其他药物或药用辅料联用实现对铁死亡相关疾病的治疗。
与现有技术方案相比,本发明至少具有以下有益效果:
本发明提供了富马酸酯及其可药用盐的新应用,所述的新应用为富马酸酯及其可药用盐在制备铁死亡相关疾病的药物中的应用,为研究铁死亡疾病的治疗策略提供了理论依据,为制备新的铁死亡相关疾病的药物提供了一个嵌入点。
附图说明
图1是富马酸二甲酯对HepG2细胞的GSH水平、GSSH水平的影响结果图;
图2是富马酸单甲酯对HepG2细胞的GSH水平、GSSH水平的影响结果图;
图3是富马酸二甲酯和富马酸单甲酯对由乙醇引起的细胞膜脂质过氧化水平的影响结果图;
图4是富马酸二甲酯和富马酸单甲酯对由DOX引起的细胞膜脂质过氧化水平上调的影响结果图;
图5是富马酸二甲酯和富马酸单甲酯对由乙醇引起的细胞GPX4蛋白水平下调的影响结果图;
图6是富马酸二甲酯和富马酸单甲酯对由DOX引起的细胞GPX4蛋白水平下调的影响结果图;
图7是小鼠肝脏组织H&E染色病理结果图,a、b、c、d图分别为对照组、模型组、模型+低剂量富马酸二甲酯组和模型+高剂量富马酸二甲酯组;
图8是小鼠肝脏组织anti-GPX4免疫组化病理结果图,a、b、c、d图分别为对照组、模型组、模型+低剂量富马酸二甲酯组和模型+高剂量富马酸二甲酯组;
图9是小鼠肝脏组织anti-4-HNE免疫组化病理结果图,a、b、c、d图分别为对照组、模型组、模型+低剂量富马酸二甲酯组和模型+高剂量富马酸二甲酯组;
图10是小鼠肝脏组织Western Blot检测GPX4蛋白水平结果图,a、b、c、d图分别为对照组、模型组、模型+低剂量富马酸二甲酯组和模型+高剂量富马酸二甲酯组;
图11是大鼠肝脏组织H&E染色病理结果图,a、b、c、d图分别为对照组、模型组、模型+低剂量富马酸二甲酯组和模型+高剂量富马酸二甲酯组;
图12是大鼠肝脏组织anti-GPX4免疫组化病理结果图,a、b、c、d图分别为对照组、模型组、模型+低剂量富马酸二甲酯组和模型+高剂量富马酸二甲酯组;
图13是大鼠肝脏组织anti-4-HNE免疫组化病理结果图,a、b、c、d图分别为对照组、模型组、模型+低剂量富马酸二甲酯组和模型+高剂量富马酸二甲酯组;
图14是大鼠肝脏组织天狼星红染色病理结果图,a、b、c、d图分别为对照组、模型组、模型+低剂量富马酸二甲酯组和模型+高剂量富马酸二甲酯组。
具体实施方式
下面通过具体实施方式来进一步说明本发明的技术方案。本领域技术人员应该明了,所述实施例仅仅是帮助理解本发明,不应视为对本发明的具体限制。
以下试验所用材料包括:
富马酸二甲酯(Sigma-Aldric,242926;10μM),富马酸单甲酯(Sigma-Aldrich,651419;10μM),乙醇(Sigma-Aldrich,E7023;80mM),多柔比星(Solarbio,D8740;10μM),铁死亡抑制剂Ferrostatin-1(Sigma-Aldric,SML0583;1μM),铁死亡激活剂Erastin(Selleck,S7242;10μM),NRF2抗体(Proteintech,16396-1-AP;1:1000),GXP4抗体(Abcam,ab125066;1:1000),ACTB/β-actin抗体(Proteintech,20536-1-AP;1:1000),GXP4抗体(Abcam,ab125066;1:100),4-HNE抗体(Abcam,ab46545;1:100);
人肝细胞HepG2和LO2细胞从湘雅医院细胞库获得,培养基为含10%胎牛血清(Gibco,10091148)以及100U/mL氨苄青霉素+链霉素双抗(Gibco,10378016)的DMEM培养基(HyClone,SH30022.01)和RPMI培养基(HyClone,SH30809.01),细胞培养箱条件为37℃恒温恒湿,并恒定二氧化碳浓度为5%。
实施例1
体外抑制肝细胞铁死亡试验:
(1)GSH和GSSG水平测定试验:
单层贴壁HepG2细胞用胰蛋白酶消化成单细胞悬液,于96孔板4000个/孔进行细胞铺板。4-6h细胞贴壁后,向细胞培养板中分别加入DMSO、10μM的富马酸二甲酯、30μM的富马酸二甲酯、10μM的富马酸单甲酯、30μM的富马酸单甲酯,孵育20h,利用GSH/GSSG-GloTMAssay试剂盒(Promega,V6611)进行细胞总GSH和GSSH检测,具体操作按说明书进行,试验重复进行三次。结果如图1和图2所示。
由图中结果可知:富马酸二甲酯和富马酸单甲酯能够显著上调HepG2细胞的GSH水平和GSSH水平,且具有浓度依赖性,随富马酸酯浓度的上升其上调水平也上升(说明书附图中的富马酸二甲酯和富马酸单甲酯分别用DMF和MMF表示)。
(2)细胞膜脂质过氧化水平测定试验:
向单层贴壁肝HepG2和LO2细胞,分别加入DMSO、1μM的Erastin、80mM的乙醇、10μM的DOX、10μM的DOX+10μM的富马酸二甲酯、10μM的DOX+10μM的富马酸单甲酯、10μM的DOX+1μM的Ferrostatin-1、80mM的乙醇+10μM的富马酸二甲酯、80mM的乙醇+10μM的富马酸单甲酯、80mM的乙醇+1μM的Ferrostatin-1,孵育6h后,用4℃预冷PBS缓冲液轻柔冲洗细胞3次,每100万个细胞使用0.1mL IP裂解液(Beyotime,P0013)进行匀浆,4℃离心后收集上清。通过MDA检测试剂盒(Beyotime,S0131)测定细胞膜脂质过氧化水平,具体操作按说明书进行,试验重复进行三次。结果如图3和图4所示。
由图中结果可知:乙醇和DOX的刺激能显著上调HepG2和LO2细胞膜的脂质过氧化水平,说明乙醇和DOX会引起细胞铁死亡的发生。富马酸二甲酯和富马酸单甲酯能显著抑制由乙醇和DOX引起的细胞膜脂质过氧化水平,说明富马酸二甲酯和富马酸单甲酯能在体外水平抑制细胞铁死亡的发生(说明书附图中的富马酸二甲酯和富马酸单甲酯分别用DMF和MMF表示)。
(3)Western Blot试验1:
向单层贴壁肝HepG2和LO2细胞,分别加入80mM的乙醇、80mM的乙醇+10μM的富马酸二甲酯、80mM的乙醇+10μM的富马酸单甲酯、80mM的乙醇+1μM的Ferrostatin-1,孵育6h后,用4℃预冷PBS缓冲液轻柔冲洗细胞3次,再用RIPA裂解液(Beyotime,P0013B)提取细胞总蛋白,具体操作按说明书进行。提取好的蛋白通过BCA蛋白浓度测定试剂盒(Beyotime,P0010S)测定浓度后,-20℃保存备用。将各组蛋白样品和2×Loading Buffer按体积比1:1配制,置于100℃金属浴中煮沸变性10min,随后冰上放置冷却5min,等待上样,具体操作按说明书进行,试验重复进行三次。结果如图5所示。
由图中结果可知:乙醇的刺激能显著下调HepG2和LO2细胞的GPX4蛋白水平,说明乙醇会引起细胞铁死亡的发生。富马酸二甲酯和富马酸单甲酯能显著上调细胞的GPX4蛋白水平,说明富马酸二甲酯和富马酸单甲酯能在体外水平抑制细胞铁死亡的发生(说明书附图中的富马酸二甲酯和富马酸单甲酯分别用DMF和MMF表示)。
(4)Western Blot试验2:
向单层贴壁肝HepG2和LO2细胞,分别加入10μM的DOX、10μM的DOX+10μM的富马酸二甲酯、10μM的DOX+10μM的富马酸单甲酯、10μM的DOX+1μM的Ferrostatin-1,孵育6h后,用4℃预冷PBS缓冲液轻柔冲洗细胞3次,再用RIPA裂解液(Beyotime,P0013B)提取细胞总蛋白,具体操作按说明书进行。提取好的蛋白通过BCA蛋白浓度测定试剂盒(Beyotime,P0010S)测定浓度后,-20℃保存备用。将各组蛋白样品和2×Loading Buffer按体积比1:1配制,置于100℃金属浴中煮沸变性10min,随后冰上放置冷却5min,等待上样,具体操作按说明书进行,试验重复进行三次。结果如图6所示。
由图中结果可知:DOX的刺激能显著下调HepG2和LO2细胞的GPX4蛋白水平,说明DOX会引起细胞铁死亡的发生。富马酸二甲酯和富马酸单甲酯能显著上调细胞的GPX4蛋白水平,说明富马酸二甲酯和富马酸单甲酯能在体外水平抑制细胞铁死亡的发生(说明书附图中的富马酸二甲酯和富马酸单甲酯分别用DMF和MMF表示)。
实施例2
对酒精性脂肪性肝病模型小鼠肝脏发生铁死亡的抑制试验:
将24只8周龄SPF级C57BL/6雄性小鼠随机分成4组,分别为空白组、模型组、模型+低剂量富马酸二甲酯(100mg/Kg)组和模型+高剂量富马酸二甲酯(200mg/Kg)组。小鼠最初自由饮用Lieber-DeCarli对照饮食5天来使他们逐渐适应液体饮食和管饲,随后,模型组允许自由饮用含有5%(体积/体积)标准Lieber-DeCarli酒***体饲料10天,而对照组用等热量对照饮食。从第11天开始,模型+给药组分别给予富马酸二甲酯(100mg/Kg)和富马酸二甲酯(200mg/Kg)每日一次灌胃给药,连续10天。之后用5%水合氯醛将小鼠腹腔麻醉,收集肝脏组织,部分于10%***中固定,部分于液氮中保存。再分别进行如下试验(说明书附图中的富马酸二甲酯用DMF表示):
(1)小鼠肝脏组织H&E染色病理结果观察:
具体方法为:将在10%***中固定好的小鼠肝脏组织标本进行脱水、浸蜡和包埋,将包埋好的石蜡组织切片。然后将组织切片在65℃恒温烤箱中烘烤60min溶解石蜡,再依次放入二甲苯中脱蜡透明,浓度由高到低的的乙醇溶液中水化。PBS溶液冲洗组织切片后,用苏木素染色10min,1%盐酸酒精分化。在流水返蓝后,伊红染色3min,最后放入浓度由低到高的乙醇溶液中脱水,二甲苯透明,中性树胶封片。在常温下干燥后,通过显微镜下采集图像并进行数据分析。
结果如图7所示,图a为正常对照组,图b为酒精性肝病模型组,图c为酒精性肝病DMF(100mg/Kg)治疗组,图d为酒精性肝病DMF(200mg/Kg)治疗组。
(2)小鼠肝脏组织anti-GPX4免疫组化病理检测试验:
具体方法为:将石蜡包埋的组织切片在65℃恒温烤箱中烘烤60min溶解石蜡,再依次放入二甲苯中脱蜡透明,浓度由高到低的的乙醇溶液中水化。PBS溶液冲洗组织切片后,分别滴加即用型免疫组化超敏UltraSensitiveTM SP试剂盒(迈新生物,KIT-9707)中试剂A去除组织内源性过氧化物酶,试剂B羊血清封闭,在组织切片上滴加适当浓度的anti-GPX4一抗溶液,4℃过夜。第二日,取出切片,常温放置30min后,依次滴加试剂C(二抗溶液,注意来源与一抗相同)和试剂D,具体操作按说明书进行。然后DAB显色,苏木素染色,1%盐酸酒精分化。在流水返蓝后,放入浓度由低到高的乙醇溶液中脱水,二甲苯透明,中性树胶封片。在常温下干燥后,通过显微镜下采集图像并进行数据分析。
结果如图8所示,图a为正常对照组,图b为酒精性肝病模型对照组,图c为酒精性肝病DMF(100mg/Kg)治疗组,图d为酒精性肝病DMF(200mg/Kg)治疗组。
(3)小鼠肝脏组织anti-4-HNE免疫组化病理检测试验:
具体方法为:将石蜡包埋的组织切片在65℃恒温烤箱中烘烤60min溶解石蜡,再依次放入二甲苯中脱蜡透明,浓度由高到低的的乙醇溶液中水化。PBS溶液冲洗组织切片后,分别滴加即用型免疫组化超敏UltraSensitiveTM SP试剂盒(迈新生物,KIT-9707)中试剂A去除组织内源性过氧化物酶,试剂B羊血清封闭,在组织切片上滴加适当浓度的anti-HNE一抗溶液,4℃过夜。第二日,取出切片,常温放置30min后,依次滴加试剂C(二抗溶液,注意来源与一抗相同)和试剂D,具体操作按说明书进行。然后DAB显色,苏木素染色,1%盐酸酒精分化。在流水返蓝后,放入浓度由低到高的乙醇溶液中脱水,二甲苯透明,中性树胶封片。在常温下干燥后,通过显微镜下采集图像并进行数据分析。
结果如图9所示,图a为正常对照组,图b为酒精性肝病模型对照组,图c为酒精性肝病DMF(100mg/Kg)治疗组,图d为酒精性肝病DMF(200mg/Kg)治疗组。
(4)小鼠肝脏组织Western Blot检测GPX4蛋白水平试验:
具体方法为:取出在液氮中保存的小鼠肝脏标本,用0.5%NP-40裂解液提取蛋白,-20℃保存备用。将各组蛋白样品和2×Loading Buffer按体积比1:1配制,置于100℃金属浴中煮沸变性10min,再冰上放置冷却5min后上样。依次进行15%SDS-PAGE凝胶电泳,转膜,一抗二抗免疫反应,化学发光显影与凝胶图象分析,具体操作按说明书进行,试验重复进行三次。
结果如图10所示。
由图7-10中结果可知:酒精性肝病模型组肝脏组织内存在大量脂滴,肝细胞中度脂肪变性,伴有少数肝细胞气球样变性及点状坏死、中央静脉周围炎,GPX4蛋白水平显著下调,4-HNE蛋白水平显著上调,提示体内水平铁死亡的发生。在对模型小鼠给予富马酸二甲酯(100mg/Kg或200mg/Kg)每日一次灌胃,连续给药10天后,观察到肝脏细胞脂肪变性和中央静脉周围炎症明显减轻,甚至趋于正常对照组肝脏组织,且GPX4蛋白水平显著上调,4-HNE蛋白水平显著下调,说明富马酸二甲酯在体内水平能够明显抑制铁死亡。
实施例3
对肝纤维化模型大鼠肝脏发生铁死亡的抑制试验:
将36只7周龄200-250g的SPF级SD大鼠随机分为4组,分别为对照组、模型组、模型+低剂量富马酸二甲酯(15mg/Kg)组和模型+高剂量富马酸二甲酯(25mg/Kg)组。模型组均采用皮下注射40%CCl4花生油溶液0.3ml/100g进行造模,每周2次。第二周开始,以10%酒精为唯一饮料,饲以混有0.5%胆固醇的玉米粉饲料,连续造模8周。造模同时,模型+给药组分别同时给予富马酸二甲酯(15mg/Kg)和富马酸二甲酯(25mg/Kg)每日一次灌胃给药,连续8周。之后用5%水合氯醛将小鼠腹腔麻醉后,收集肝脏组织,部分于***中固定,部分于液氮中保存。再分别进行如下试验(说明书附图中的富马酸二甲酯用DMF表示):
(1)大鼠肝脏组织H&E染色病理结果观察:
具体方法为:将在10%***中固定好的大鼠肝脏组织标本进行脱水、浸蜡和包埋,将包埋好的石蜡组织切片。然后将组织切片在65℃恒温烤箱中烘烤60min溶解石蜡,再依次放入二甲苯中脱蜡透明,浓度由高到低的的乙醇溶液中水化。PBS溶液冲洗组织切片后,用苏木素染色10min,1%盐酸酒精分化。在流水返蓝后,伊红染色3min,最后放入浓度由低到高的乙醇溶液中脱水,二甲苯透明,中性树胶封片。在常温下干燥后,通过显微镜下采集图像并进行数据分析。
结果如图11所示,图a为正常对照组,图b为肝纤维化模型对照组,图c为肝纤维化DMF(15mg/Kg)治疗组,图d为肝纤维化DMF(25mg/Kg)治疗组。
(2)大鼠肝脏组织anti-GPX4免疫组化病理检测试验:
具体方法为:将石蜡包埋的组织切片在65℃恒温烤箱中烘烤60min溶解石蜡,再依次放入二甲苯中脱蜡透明,浓度由高到低的的乙醇溶液中水化。PBS溶液冲洗组织切片后,分别滴加即用型免疫组化超敏UltraSensitiveTM SP试剂盒(迈新生物,KIT-9707)中试剂A去除组织内源性过氧化物酶,试剂B羊血清封闭,在组织切片上滴加适当浓度的anti-GPX4一抗溶液,4℃过夜。第二日,取出切片,常温放置30min后,依次滴加试剂C(二抗溶液,注意来源与一抗相同)和试剂D,具体操作按说明书进行。然后DAB显色,苏木素染色,1%盐酸酒精分化。在流水返蓝后,放入浓度由低到高的乙醇溶液中脱水,二甲苯透明,中性树胶封片。在常温下干燥后,通过显微镜下采集图像并进行数据分析。
结果如图12所示,图a为正常对照组,图b为肝纤维化模型对照组,图c为肝纤维化DMF(15mg/Kg)治疗组,图d为肝纤维化DMF(25mg/Kg)治疗组。
(3)大鼠肝脏组织anti-4-HNE免疫组化病理检测试验:
具体方法为:将石蜡包埋的组织切片在65℃恒温烤箱中烘烤60min溶解石蜡,再依次放入二甲苯中脱蜡透明,浓度由高到低的的乙醇溶液中水化。PBS溶液冲洗组织切片后,分别滴加即用型免疫组化超敏UltraSensitiveTM SP试剂盒(迈新生物,KIT-9707)中试剂A去除组织内源性过氧化物酶,试剂B羊血清封闭,在组织切片上滴加适当浓度的anti-HNE一抗溶液,4℃过夜。第二日,取出切片,常温放置30min后,依次滴加试剂C(二抗溶液,注意来源与一抗相同)和试剂D,具体操作按说明书进行。然后DAB显色,苏木素染色,1%盐酸酒精分化。在流水返蓝后,放入浓度由低到高的乙醇溶液中脱水,二甲苯透明,中性树胶封片。在常温下干燥后,通过显微镜下采集图像并进行数据分析。
结果如图13所示,图a为正常对照组,图b为肝纤维化模型对照组,图c为肝纤维化DMF(15mg/Kg)治疗组,图d为肝纤维化DMF(25mg/Kg)治疗组。
(4)大鼠肝脏组织天狼星红染色病理结果观察:
具体方法为:利用天狼星红染色试剂盒(Solarbio,G1470),取大鼠肝脏组织固定于10%***固定液8-12h,常规脱水包埋,切片,厚度6μm左右,常规脱蜡至水。Weigert铁苏木素染色液染色10-20min,盐酸酒精分化数秒。流水返蓝15min,ddH2O洗一次。天狼星红染色液滴染1h,流水稍微清洗,常规脱水透明,中性树胶封片,进行观察。
结果如图14所示,图a为正常对照组,图b为肝纤维化模型对照组,图c为肝纤维化DMF(15mg/Kg)治疗组,图d为肝纤维化DMF(25mg/Kg)治疗组。
由图11-14中结果可知:纤维化模型组大鼠肝脏组织内存在大量脂滴,肝细胞中度脂肪变性,伴有少数肝细胞气球样变性及点状坏死,汇管区周围炎症,纤维化扩大,局限性窦周及小叶内纤维化,提示肝脏发生轻度纤维化,GPX4蛋白水平显著下调,4-HNE蛋白水平显著上调,提示纤维化模型组大鼠肝脏组织发生铁死亡。对部分模型小鼠,在造模的同时,给予富马酸二甲酯(15mg/Kg或25mg/Kg)每日一次灌胃,连续给药8周后,观察到上述病理改变明显减轻,甚至趋于正常对照组的肝脏组织。且GPX4蛋白水平显著上调,4-HNE蛋白水平显著下调,说明富马酸二甲酯在体内水平能够明显抑制铁死亡的发生。
申请人声明,本发明通过上述实施例来说明本发明的富马酸酯及其可药用盐在制备治疗铁死亡相关疾病的药物中的应用,但本发明并不局限于上述实施例,即不意味着本发明必须依赖上述实施例才能实施。所属技术领域的技术人员应该明了,对本发明的任何改进,对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等,均落在本发明的保护范围和公开范围之内。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于上述实施方式中的具体细节,在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,这些简单变型均属于本发明的保护范围。
另外需要说明的是,在上述具体实施方式中所描述的各个具体技术特征,在不矛盾的情况下,可以通过任何合适的方式进行组合,为了避免不必要的重复,本发明对各种可能的组合方式不再另行说明。
Claims (10)
1.富马酸酯及其可药用盐在制备治疗铁死亡相关疾病的药物中的应用。
2.如权利要求1所述的应用,其特征在于,所述富马酸酯包括富马酸二甲酯和/或富马酸单甲酯。
3.如权利要求1或2所述的应用,其特征在于,所述药物的剂型包括片剂、散剂、颗粒剂、胶囊剂、注射剂、喷雾剂、膜剂、栓剂、滴鼻剂或滴丸剂。
4.如权利要求1-3中任一项所述的应用,其特征在于,所述药物的给药途径包括静脉注射、腹腔注射、肌肉注射、皮下注射、口服给药、舌下给药、鼻腔给药或经皮给药。
5.如权利要求1-4中任一项所述的应用,其特征在于,所述铁死亡相关疾病包括肝脏疾病、神经***疾病、肿瘤、急性肾衰竭、缺血再灌注损伤或铁代谢相关疾病。
6.如权利要求5中任一项所述的应用,其特征在于,所述肝脏疾病是由铁死亡相关因子水平异常引起的肝脏疾病;
优选地,所述肝脏疾病包括血色沉着病、原发性胆汁性胆管炎、肝纤维化。
7.如权利要求5或6所述的应用,其特征在于,所述神经***疾病包括帕金森病、阿尔兹海默症、脑室周围白质软化症、亨廷顿病、运动神经衰退症或脑外伤;
优选地,所述肿瘤包括头颈部恶性肿瘤、乳腺癌、食管癌、肝癌、胰腺癌、肾癌或弥漫性大B细胞淋巴瘤。
8.如权利要求5-7中任一项所述的应用,其特征在于,所述缺血再灌注损伤包括心肌缺血再灌注损伤、肝缺血再灌注损伤或肾缺血再灌注损伤;
优选地,所述铁代谢相关疾病包括动脉粥样硬化或糖尿病。
9.如权利要求1-8中任一项所述的应用,其特征在于,所述富马酸酯及其可药用盐为负载于药用载体上的富马酸酯及其可药用盐。
10.如权利要求1-8中任一项所述的应用,其特征在于,所述富马酸酯及其可药用盐为药物组合物中含有的富马酸酯及其可药用盐。
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PCT/CN2019/092233 WO2020113942A1 (zh) | 2018-12-05 | 2019-06-21 | 富马酸酯及其可药用盐在制备治疗铁死亡相关疾病的药物中的应用 |
US17/335,202 US20210283089A1 (en) | 2018-12-05 | 2021-06-01 | Use of fumaric acid esters and pharmaceutically acceptable salts thereof in preparing pharmaceuticals for treating ferroptosis-related diseases |
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CN113186269A (zh) * | 2021-04-07 | 2021-07-30 | 宁夏医科大学 | 铁死亡参与小鼠动脉粥硬化及泡沫细胞形成过程的研究方法 |
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CN113186269A (zh) * | 2021-04-07 | 2021-07-30 | 宁夏医科大学 | 铁死亡参与小鼠动脉粥硬化及泡沫细胞形成过程的研究方法 |
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