CN117771246A - RPN11抑制剂Capzimin在制备治疗非酒精性脂肪性肝炎的药物中的应用 - Google Patents
RPN11抑制剂Capzimin在制备治疗非酒精性脂肪性肝炎的药物中的应用 Download PDFInfo
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Abstract
本发明公开了RPN11抑制剂Capzimin在制备治疗非酒精性脂肪性肝炎的药物中的应用。本发明通过在高脂高胆固醇饮食喂养的成年NASH小鼠模型中进行体内实验,首次证实了RPN11抑制剂Capzimin可以显著改善NASH相关症状,包括降低肝脏甘油三酯沉积、缓解肝脏损伤、改善肝脏炎症和纤维化,因此,本发明为有效治疗非酒精性脂肪性肝炎提供了一种新的药物,本发明提出的RPN11抑制剂Capzimin作为治疗非酒精性脂肪性肝炎的药物显示出潜在的临床应用前景和应用价值。
Description
技术领域
本发明涉及RPN11抑制剂Capzimin在制备治疗非酒精性脂肪性肝炎的药物中的应用,属于生物医药技术领域。
背景技术
非酒精性脂肪性肝炎(Nonalcoholic steatohepatitis,NASH)已成为全球性重大慢性肝病和代谢性疾病。NASH主要临床特征包括肝脏甘油三酯沉积、慢性炎症和持续性肝脏损伤。此外,伴随着纤维化的发生,NASH可发展为肝硬化,成为肝癌、肝衰竭等终末期肝病的主要原因之一。然而截止目前,临床上仍缺乏有效而特异的治疗手段,使得NASH成为严重威胁国人身心健康的疾病。
RPN11属于去泛素化酶家族,通过去除靶蛋白的泛素化修饰、增加靶蛋白的稳定性而发挥作用。既往研究开发了RPN11特异性抑制剂Capzimin,发现具有抑制肿瘤细胞生长的作用。但Capzimin能否作为NASH的治疗药物,国内外尚无报道。
发明内容
本发明的目的是:提供RPN11抑制剂Capzimin在制备治疗非酒精性脂肪性肝炎的药物中的应用。本发明在高脂高胆固醇饮食喂养的成年NASH小鼠中,通过腹腔注射Capzimin,发现NASH相关症状得以明显缓解,包括肝脏重量降低,肝脏脂质含量降低,肝脏炎症和纤维化程度降低。
为了实现上述目的,本发明提供RPN11抑制剂Capzimin在制备治疗非酒精性脂肪性肝炎的药物中的应用,所述RPN11抑制剂Capzimin的CAS号为:2084868-04-0,化学结构式如下所示:
优选地,所述药物包括医学上可接受的载体和有效量的活性成分,所述活性成分为Capzimin或其药学上可接受的盐形式。
优选地,所述药物的剂型包括注射剂、片剂、粉剂、混悬剂、胶囊剂、丸剂或糖浆。
优选地,所述药物通过降低肝脏重量、降低肝脏和血浆中的甘油三酯含量、降低血浆丙氨酸转氨酶和天冬氨酸转氨酶含量、改善肝脏炎症和肝脏胶原纤维沉积改善非酒精性脂肪性肝炎。
本发明还提供了RPN11抑制剂Capzimin在制备降低肝脏和血浆中的甘油三酯含量的药物中的应用,所述RPN11抑制剂Capzimin的CAS号为:2084868-04-0,化学结构式如下所示:
本发明还提供了RPN11抑制剂Capzimin在制备治疗肝脏损伤的药物中的应用,所述RPN11抑制剂Capzimin的CAS号为:2084868-04-0,化学结构式如下所示:
本发明还提供了RPN11抑制剂Capzimin在制备改善肝脏炎症和肝脏胶原纤维沉积的药物中的应用,所述RPN11抑制剂Capzimin的CAS号为:2084868-04-0,化学结构式如下所示:
本发明与现有技术相比,具有如下有益效果:
本发明在高脂高胆固醇饮食喂养的成年NASH小鼠模型中,通过使用RPN11抑制剂Capzimin,可以显著改善NASH相关症状,包括降低肝脏重量,降低肝脏和血浆甘油三酯含量,降低肝脏炎症和纤维化程度;因此,本发明为临床上治疗非酒精性脂肪性肝炎(NASH)提供了一种新的治疗药物——RPN11抑制剂Capzimin。
附图说明
图1:NASH小鼠肝脏组织RPN11的mRNA表达增加(A),蛋白表达增加(B);
图2:Capzimin显著降低NASH小鼠的肝脏重量(A),肝脏重量和体重比值(B),肝脏甘油三酯含量(C)和血浆甘油三酯含量(D);
图3:Capzimin显著降低NASH小鼠的血浆谷丙转氨酶含量(A),血浆谷草转氨酶含量(B);
图4:Capzimin显著改善NASH小鼠的肝脏气球样变性和脂质沉积;
图5:Capzimin显著减少NASH小鼠的肝脏炎性细胞浸润(A,B)。A:F4/80免疫组化染色,表示炎性细胞浸润和激活。B:两组小鼠F4/80染色的统计学分析;
图6:Capzimin显著减少NASH小鼠的肝脏胶原纤维沉积;A:天狼猩红染色,表示胶原纤维沉积;B:两组小鼠肝脏天狼猩红染色的统计学分析;
图7:Capzimin显著减少NASH小鼠肝脏中炎症相关基因的mRNA表达,显著减少NASH小鼠肝脏中纤维化相关基因的mRNA表达。
具体实施方式
为使本发明更明显易懂,兹以优选实施例,并配合附图作详细说明如下。
下述实施例中所使用的试验方法如无特殊说明,均为常规方法;所使用的材料、试剂等,如无特殊说明,为可从商业途径得到的试剂和材料。
本发明以下实施例中涉及的实验材料与实验方法如下:
1.8周龄C57BL/6雄性小鼠购自上海斯莱克实验动物有限公司,小鼠饲养和实验在上海交通大学医学院实验动物科学部完成。正常饮食饲料购自上海斯莱克实验动物有限公司,货号M01-F,成分包括:10Kcal%脂肪,70Kcal%碳水化合物,20Kcal%蛋白质。高脂高胆固醇饮食饲料购自美国Research Diet公司,货号D09100310,成分包括:40Kcal%脂肪,2%胆固醇,20Kcal%果糖。所有小鼠在到达实验终点后,通过安乐死处理,全血经抗凝、离心后,取上清,获得血浆,置于-80度保存。后续实验中,通过试剂盒检测血浆甘油三酯含量。留取部分新鲜肝脏组织,液氮速冻后,置于-80度保存,后续实验中,在NP40裂解液中高温裂解(95摄氏度),离心后获得上清液,通过试剂盒检测甘油三酯含量;部分冻存的肝脏组织用于抽提RNA,开展实时定量PCR实验和蛋白质免疫印迹,检测目的基因的mRNA和蛋白表达丰度。留取部分新鲜肝脏组织,置于4%多聚甲醛中,固定48小时后,制作石蜡切片,用于伊红苏木精染色;制作冰冻切片,用于油红O染色。
2.甘油三酯含量检测:按照英国Abcam公司提供的甘油三酯定量检测试剂盒的说明书完成检测,货号ab65336。
3.血浆丙氨酸转氨酶、天冬氨酸转氨酶的检测:分别按照中国上海科华公司提供的定量检测试剂盒的说明书完成检测。
4.F4/80免疫荧光染色:F4/80是炎症细胞的标志性蛋白,通过对肝脏石蜡切片开展免疫荧光染色,从而观察到肝脏中炎症细胞的激活和浸润程度。F4/80抗体购自英国Abcam公司,货号ab6640。
5.天狼猩红染色:天狼猩红是一种酸性染料,与呈碱性的胶原可发生反应而显色,从而观察到肝脏中胶原纤维的沉积。天狼猩红染料购自上海瑞雨生物科技有限公司,货号Bry-0013。
6.肝脏基因的表达检测:通过实时定量PCR实验,检测下列基因的表达:RPN11、肿瘤坏死因子(Tumor necrosis factor,TNF)、白介素1beta(Interleukin 1beta,IL1b)、白介素6(Interleukin 6,IL6)、白介素12beta(Interleukin 12beta,IL12b)、诱导型一氧化氮合酶(Nitric oxide synthase 2,inducible,Nos2)、趋化因子配体2[Chemokine(C-Cmotif)ligand 2,CCL2]、趋化因子配体5[Chemokine(C-C motif)ligand 5,CCL5]和粘附G蛋白偶联受体E1(Adhesion G protein-coupled receptor E1,Adgre1)是肝脏炎症的标志性基因。1型胶原纤维(Collagen,type I,alpha 1,Col1a1;Collagen,type I,alpha 2,Col1a2)、平滑肌细胞特异性alpha-肌动蛋白(Actin alpha 2,smooth muscle,Acta2)、转化生长因子beta 1(Transforming growth factor,beta 1,TGFb1)和基质金属蛋白酶13(Matrix metallopeptidase 13,MMP13)是肝脏纤维化的标志性基因。36B4基因作为实时定量PCR实验的内参。引物序列如下所示。
实时定量PCR实验的引物:
小鼠RPN11:
Forward:5’-GGAGGTGGTGATAGCCGGTAT-3’(SEQ ID NO:1);
Reverse:5’-TGGGTAATCCATAGAGCCCAG-3’(SEQ ID NO:2);
小鼠36B4:
Forward:5’-AGATTCGGGATATGCTGTTGGC-3’(SEQ ID NO:3);
Reverse:5’-TCGGGTCCTAGACCAGTGTTC-3’(SEQ ID NO:4);
小鼠TNF:
Forward:5’-CAGGCGGTGCCTATGTCTC-3’(SEQ ID NO:5);
Reverse:5’-CGATCACCCCGAAGTTCAGTAG-3’(SEQ ID NO:6);
小鼠IL1b:
Forward:5’-GAAATGCCACCTTTTGACAGTG-3’(SEQ ID NO:7);Reverse:5’-TGGATGCTCTCATCAGGACAG-3’(SEQ ID NO:8);
小鼠IL6:
Forward:5’-TAGTCCTTCCTACCCCAATTTCC-3’(SEQ ID NO:9);Reverse:5’-TTGGTCCTTAGCCACTCCTTC-3’(SEQ ID NO:10);
小鼠IL12b:
Forward:5’-TGGTTTGCCATCGTTTTGCTG-3’(SEQ ID NO:11);
Reverse:5’-ACAGGTGAGGTTCACTGTTTCT-3’(SEQ ID NO:12);小鼠Nos2:
Forward:5’-CGGCAAACATGACTTCAGGC-3’(SEQ ID NO:13);
Reverse:5’-TAGGTCGATGCACAACTGGG-3’(SEQ ID NO:14);
小鼠CCL2:
Forward:5’-TTAAAAACCTGGATCGGAACCAA-3’(SEQ ID NO:15);Reverse:5’-GCATTAGCTTCAGATTTACGGGT-3’(SEQ ID NO:16);小鼠CCL5:
Forward:5’-GCTGCTTTGCCTACCTCTCC-3’(SEQ ID NO:17);
Reverse:5’-TCGAGTGACAAACACGACTGC-3’(SEQ ID NO:18);小鼠Adgre1:
Forward:5’-CCCCAGTGTCCTTACAGAGTG-3’(SEQ ID NO:19);Reverse:5’-GTGCCCAGAGTGGATGTCT-3’(SEQ ID NO:20);
小鼠Col1a1:
Forward:5’-GCTCCTCTTAGGGGCCACT-3’(SEQ ID NO:21);
Reverse:5’-CCACGTCTCACCATTGGGG-3’(SEQ ID NO:22);
小鼠Acta2:
Forward:5’-GTCCCAGACATCAGGGAGTAA-3’(SEQ ID NO:23);Reverse:5’-TCGGATACTTCAGCGTCAGGA-3’(SEQ ID NO:24);
小鼠TGFb1:
Forward:5’-CTCCCGTGGCTTCTAGTGC-3’(SEQ ID NO:25);
Reverse:5’-GCCTTAGTTTGGACAGGATCTG-3’(SEQ ID NO:26);
小鼠MMP13:
Forward:5’-GGAGCCCTGATGTTTCCCAT-3’(SEQ ID NO:27);
Reverse:5’-GTCTTCATCGCCTGGACCATA-3’(SEQ ID NO:28);
7.RPN11蛋白表达的检测:通过蛋白质免疫印迹,检测RPN11蛋白表达,RPN11抗体购自Proteintech公司,货号12059-1-AP;b-Actin蛋白作为内参,抗体购自Santa Cruz公司,货号sc-47778。
8.RPN11抑制剂Capzimin为常规商购试剂,其CAS号为:2084868-04-0,化学结构式如下所示:
9.统计分析:本研究中数值变量均以均数+标准误表示,采用双尾Student-t检验比较两组间的变量。当P<0.05时,认为两组间具有统计学差异。*表示P<0.05,**表示P<0.01,***表示P<0.001。
实施例1
NASH小鼠肝脏组织RPN11表达增加:
高脂高胆固醇饮食(High-fat high-cholesterol diet,简称HFHC饮食)喂养C57BL/6小鼠是构建NASH动物模型的常用方法。将8周龄C57BL/6雄性小鼠随机分为两组,每组6只,分别喂养正常饮食饲料或HFHC饮食饲料。24周后,安乐死小鼠,取两组肝脏组织,通过实时定量PCR实验(Quantitative real-time PCR)和蛋白质免疫印迹实验(WesternBlots),检测基因表达。结果显示,同正常饮食组小鼠相比,HFHC饮食喂养小鼠肝脏RPN11的messenger RNA(mRNA)和蛋白表达均显著增加(图1A和1B)。该结果表明,NASH小鼠模型肝脏组织RPN11表达增加。
实施例2
RPN11抑制剂Capzimin改善NASH:
实施例1的结果提示,抑制RPN11可能作为改善NASH的新手段。本实施例中通过给予8周龄C57BL/6雄性小鼠喂养HFHC饮食24周,诱导NASH模型。随后,将小鼠随机分为两组,每组6只,分别给予腹腔注射RPN11抑制剂Capzimin(2.5mg/kg)或对照溶剂(玉米油),每天一次,持续2周。2周后,安乐死小鼠,取血浆和肝脏组织检测。结果表明:同对照组小鼠相比,Capzimin处理组小鼠肝脏重量、肝脏重量和体重比值、肝脏甘油三酯含量和血浆甘油三酯水平均显著降低(图2A-2D)。血浆谷丙转氨酶和谷草转氨酶均显著降低(图3A和3B),表明Capzimin能缓解肝脏损伤。伊红苏木精染色表明肝细胞气球样变性减少(图4),油红O染色表明肝脏脂质沉积减少(图4),F4/80染色表明肝脏炎性细胞浸润减少(图5A和5B),天狼猩红染色表明肝脏纤维化程度改善(图6A和6B)。基因表达检测表明,肝脏炎症和纤维化相关基因的mRNA表达显著减少(图7)。上述结果证实,Capzimin可以有效改善NASH相关症状。
上述实施例仅为本发明的优选实施例,并非对本发明任何形式上和实质上的限制,应当指出,对于本技术领域的普通技术人员,在不脱离本发明的前提下,还将可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。
Claims (7)
1.RPN11抑制剂Capzimin在制备治疗非酒精性脂肪性肝炎的药物中的应用,其特征在于,所述RPN11抑制剂Capzimin的CAS号为:2084868-04-0,化学结构式如下所示:
2.如权利要求1所述的应用,其特征在于,所述药物包括医学上可接受的载体和有效量的活性成分,所述活性成分为Capzimin或其药学上可接受的盐形式。
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注射剂、片剂、粉剂、混悬剂、胶囊剂、丸剂或糖浆。
4.如权利要求1或2所述的应用,其特征在于,所述药物通过降低肝脏重量、降低肝脏和血浆中的甘油三酯含量、降低血浆丙氨酸转氨酶和天冬氨酸转氨酶含量、改善肝脏炎症和肝脏胶原纤维沉积改善非酒精性脂肪性肝炎。。
5.RPN11抑制剂Capzimin在制备降低肝脏和血浆中的甘油三酯含量的药物中的应用,其特征在于,所述RPN11抑制剂Capzimin的CAS号为:2084868-04-0,化学结构式如下所示:
6.RPN11抑制剂Capzimin在制备治疗肝脏损伤的药物中的应用,其特征在于,所述RPN11抑制剂Capzimin的CAS号为:2084868-04-0,化学结构式如下所示:
7.RPN11抑制剂Capzimin在制备改善肝脏炎症和肝脏胶原纤维沉积的药物中的应用,其特征在于,所述RPN11抑制剂Capzimin的CAS号为:2084868-04-0,化学结构式如下所示:
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