CN116942826A - 胞质前列腺素e合酶作为预防和/或治疗非酒精性脂肪肝病的药物靶点的应用 - Google Patents
胞质前列腺素e合酶作为预防和/或治疗非酒精性脂肪肝病的药物靶点的应用 Download PDFInfo
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Abstract
本发明公开了一种胞质前列腺素E合酶作为预防和/或治疗非酒精性脂肪肝病的药物靶点的应用。本发明首次提出胞质前列腺素E合酶是非酒精性脂肪肝病的药物靶点,经实验表明,胞质前列腺素E合酶敲低可以显著降低非酒精性脂肪肝病小鼠的肝损伤,明显减轻非酒精性脂肪肝病小鼠肝脏脂质堆积;这些实验结果说明敲低胞质前列腺素E合酶对非酒精性脂肪肝病小鼠肝组织具有明显的改善作用,进而表明胞质前列腺素E合酶可以作为预防和/或治疗非酒精性脂肪肝病的靶点,为临床上非酒精性脂肪肝病药物的开发及预防或治疗提供了有价值的参考意义。
Description
技术领域
本发明涉及胞质前列腺素E合酶(cytosolic prostaglandin E synthase,cPGES)作为预防和/或治疗非酒精性脂肪肝病的药物靶点的应用,尤其涉及胞质前列腺素E合酶作为靶点在开发或制备或筛选用于预防和/或治疗非酒精性脂肪肝病的药物中的应用,属于生物医药技术领域。
背景技术
非酒精性脂肪性肝病(NAFLD)是世界范围内最常见的肝脏疾病之一。非酒精性脂肪肝病(NAFLD)的发病率和患病率在全球范围内迅速上升。据估计,全球四分之一的人口患有非酒精性脂肪性肝病。非酒精性脂肪肝炎(NASH)的发病率预计在未来10年将增加高达56%。
非酒精性脂肪肝病是一种进行性发展的肝脏疾病,良性的非酒精性脂肪肝会进一步向炎症纤维化等症状加重的非酒精性脂肪肝炎进展,最终演变为肝癌等终末期肝病,使患者走向肝移植和死亡。而目前国际上对非酒精性脂肪肝尚无公认的药物,药物治疗指南中推荐的吡格列酮具有严重的心血管系统毒性,因此探究NAFLD的发病机制,寻找安全有效的药物防治具有重要的现实意义。
发明内容
本发明的主要目的在于提供一种胞质前列腺素E合酶作为靶点在开发或制备或筛选用于预防和/或治疗非酒精性脂肪肝病的药物中的应用,以克服现有技术的不足。
本发明的另一主要目的在于提供一种胞质前列腺素E合酶或其编码基因的下调剂在制备用于预防和/或治疗非酒精性脂肪肝病的药物中的应用。
本发明的另一主要目的在于提供一种用于预防和/或治疗非酒精性脂肪肝病的药物组合物。
为实现前述发明目的,本发明采用的技术方案包括:
本发明实施例提供了胞质前列腺素E合酶作为靶点在开发或制备或筛选用于预防和/或治疗非酒精性脂肪肝病的药物中的应用。
本发明实施例还提供了胞质前列腺素E合酶或其编码基因的下调剂在制备用于预防和/或治疗非酒精性脂肪肝病的药物中的应用。
本发明实施例还提供了一种用于预防和/或治疗非酒精性脂肪肝病的药物组合物,其包括:胞质前列腺素E合酶或其编码基因的下调剂,以及药学上可接受的载体。
与现有技术相比,本发明的有益效果在于:本发明首次提出cPGES是非酒精性脂肪肝病(NAFLD)的药物靶点,经实验表明,cPGES敲低可以显著降低NAFLD小鼠的肝损伤,明显减轻NAFLD小鼠肝脏脂质堆积;这些实验结果说明敲低cPGES对NAFLD小鼠肝组织具有明显的改善作用,进而表明cPGES可以作为预防和/或治疗NAFLD的靶点,为临床上NAFLD药物的开发及预防和治疗提供了有价值的参考意义。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明中记载的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明一典型实施方案中cPGES对照组(NC)小鼠和过表达组(OE)小鼠葡萄糖耐量实验(IPGTT)结果图;
图2a-图2b为本发明一典型实施方案中cPGES对照组(NC)小鼠和过表达组(OE)小鼠肝脏组织中cPGES表达水平图;
图3a-图3b为本发明一典型实施方案中cPGES对照组(NC)小鼠和过表达组(OE)小鼠肝脏组织形态图;
图4a-图4b为本发明一典型实施方案中cPGES对照组(NC)小鼠和过表达组(OE)小鼠肝脏油红染色结果图;
图5a-图5b为本发明一典型实施方案中cPGES对照组(NC)小鼠和过表达组(OE)小鼠血清TC、TG结果图;
图6a-图6b为本发明一典型实施方案中cPGES对照组(NC)小鼠和敲低组(KD)小鼠肝脏组织中cPGES表达水平图;
图7a-图7b为本发明一典型实施方案中cPGES对照组(NC)小鼠和敲低组(KD)小鼠肝脏组织形态图;
图8a-图8b为本发明一典型实施方案中cPGES对照组(NC)小鼠和敲低组(KD)小鼠肝脏油红染色结果图;
图9a-图9b为本发明一典型实施方案中cPGES对照组(NC)小鼠和敲低组(KD)小鼠血清AST、ALT结果图。
具体实施方式
鉴于现有技术的缺陷,本案发明人经长期研究和大量实践,得以提出本发明的技术方案,下面将对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
具体的,作为本发明技术方案的一个方面,其所涉及的是胞质前列腺素E合酶作为靶点在开发或制备或筛选用于预防和/或治疗非酒精性脂肪肝病的药物中的应用。
胞质前列腺素E合酶(cPGES)是PGE2的三种合酶之一,是一种23kDa的谷胱甘肽(GSH)依赖的酶,主要存在于细胞质中。
在一些优选实施方案中,所述靶点过表达使得非酒精性脂肪肝病小鼠的葡萄糖耐受度降低。
在一些优选实施方案中,所述靶点过表达使得非酒精性脂肪肝病小鼠的肝脏组织中cPGES的表达水平升高。
在一些优选实施方案中,所述靶点过表达使得非酒精性脂肪肝病小鼠的肝脏脂质堆积。
在一些优选实施方案中,所述靶点过表达使得非酒精性脂肪肝病小鼠的血清TC或血清TG水平升高。
在一些优选实施方案中,所述靶点过表达后能够加重非酒精性脂肪肝病(NAFLD)小鼠的肝脏损伤。
在一些优选实施方案中,所述靶点敲低至少能够降低非酒精性脂肪肝病小鼠的肝脏组织中cPGES的表达水平。
在一些优选实施方案中,所述靶点敲低至少能够抑制非酒精性脂肪肝病小鼠的肝脏脂质滴堆积。
在一些优选实施方案中,所述靶点敲低至少能够降低非酒精性脂肪肝病小鼠的血清AST或血清ALT水平升高。
在一些优选实施方案中,所述非酒精性脂肪肝病包括胞质前列腺素E合酶过表达及高脂条件下诱导的非酒精性脂肪肝病。
在一些优选实施方案中,所述靶点敲低后能够改善非酒精性脂肪肝病(NAFLD)小鼠的肝组织形态损伤。
本发明实施例的另一个方面还提供了胞质前列腺素E合酶或其编码基因的下调剂在制备用于预防和/或治疗非酒精性脂肪肝病的药物中的应用。
在一些优选实施方案中,所述下调剂选自特异性干扰胞质前列腺素E合酶的编码基因表达的干扰分子、特异性抑制胞质前列腺素E合酶或其编码基因的小分子化合物,或者特异性与胞质前列腺素E合酶结合的抗体或配体。
进一步地,所述干扰分子是以胞质前列腺素E合酶的编码基因或其转录本为抑制或沉默靶标的小干扰RNA、反义核酸、微小RNA、dsRNA,或能表达或形成所述小干扰RNA、反义核酸、微小RNA、dsRNA的构建物。
本发明实施例的另一个方面还提供了一种用于预防和/或治疗非酒精性脂肪肝病的药物组合物,其包括:胞质前列腺素E合酶或其编码基因的下调剂,以及药学上可接受的载体。
在一些优选实施方案中,所述下调剂选自特异性干扰胞质前列腺素E合酶的编码基因表达的干扰分子、特异性抑制胞质前列腺素E合酶或其编码基因的小分子化合物,或者特异性与胞质前列腺素E合酶结合的抗体或配体。
进一步地,所述干扰分子是以胞质前列腺素E合酶的编码基因或其转录本为抑制或沉默靶标的小干扰RNA、反义核酸、微小RNA、dsRNA,或能表达或形成所述小干扰RNA、反义核酸、微小RNA、dsRNA的构建物。
本发明实施例的另一个方面还提供了cPGES作为靶点在制备用于预防和/或治疗个体的非酒精性脂肪性肝病的药物筛选模型中的应用。
下面结合若干优选实施例及附图对本发明的技术方案做进一步详细说明,本实施例在以发明技术方案为前提下进行实施,给出了详细的实施方式和具体的操作过程,但本发明的保护范围不限于下述的实施例。
下面所用的实施例中所采用的实验材料,如无特殊说明,均可由常规的生化试剂公司购买得到。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1 cPGES对照组(NC)小鼠和过表达组(OE)小鼠葡萄糖耐量实验(IPGTT)
每组取6只小鼠,小鼠禁食不禁水12h,12h后测量每只小鼠的空腹血糖。测量完毕后,每只小鼠腹腔注射剂量为2g/kg葡萄糖溶液,注射完葡萄糖后于15min、30min、45min、60min、90min、120min采用尾静脉取血的方式测量小鼠血糖值,所有时间段测量完毕后进行数据处理。
实施例2免疫印迹
(1)RIPA裂解液配制:临用前每10ml RIPA裂解液加入1片蛋白酶抑制剂,溶解混匀。
(2)样本制备:
动物:电子天平称量0.030g组织,每个样本加入配制好的RIPA裂解液300μl,电动匀浆器匀浆至组织破碎,置于冰上裂解30分钟,其间每10分钟涡旋一次。
(3)12000g离心15分钟,吸取上清,即为蛋白样本。
(4)配制BCA工作液:按照要求,A∶B=1∶50。现配现用,充分混匀。
(5)在96孔板中依次加入18μl双蒸水,2μl样本,200μl配制好的BCA工作液,轻轻吹打混匀后,置于37℃恒温摇床孵育20分钟,酶标仪于550nm处检测吸光度值,带入标准曲线后,计算出蛋白浓度。
(6)根据各个样本蛋白浓度,加入适量双蒸水,将各个样本调为相同蛋白浓度。再加入样本1/4体积的5×上样缓冲液,涡旋混匀后,置于金属水浴锅中,100℃煮10分钟,取出置于冰上,放置10分钟,-20℃保存备用。
(7)清洗制胶板,组装好制胶板后,检漏。根据目的蛋白分子量配制分离胶,无水乙醇压平,置于37℃至分离胶完全凝固。配制浓缩胶,根据样本数选择梳子。倒去无水乙醇,加入配制好的浓缩胶,插入梳子,避免气泡产生,置于37℃至浓缩胶完全凝固。
(8)组装电泳装置,加入适量1×电泳液,拔取梳子后,依次加入样本与蛋白marker。电泳条件:S1:70V,30分钟;S2:110V,90分钟。
(9)组装转膜装置,配制转膜液,按照蛋白marker裁胶,获得目的蛋白。按照黑胶白膜顺序将胶转入转模装置。转膜条件:80V,90分钟。
(10)转膜结束后,取出NC膜,置于封闭液中封闭1小时。用一抗稀释液按照合适比例配制一抗。将NC膜置于一抗中4℃孵育过夜。
(11)隔日,取出NC膜,TBST洗3次,每次10分钟,加入适宜的二抗(兔/鼠抗),室温孵育60分钟。TBST洗2次,每次10分钟,TBS洗10分钟。条带擦干后显影,保存图片。
实施例3 H&E染色对NAFLD肝脏组织病理学观察
具体的实验方法包括:
1、石蜡切片制备
(1)组织标本的固定:取实施例1中的各组小鼠的肾皮质组织于4%多聚甲醛中室温固定24小时,纱布包裹,标记,流水冲洗过夜;
(2)脱水与透明:经50%、60%、70%、80%和90%酒精中梯度各脱水2小时,然后于95%酒精,100%酒精-I/II/III中各脱水1小时,入二甲苯-I/II各透明30分钟;
(3)浸蜡与包埋:在58℃恒温箱中浸蜡,用石蜡-I浸蜡1.5小时,石蜡-II浸蜡2小时。放入包埋盒中60℃下进行石蜡包埋,待冷却凝固成块后将蜡块取出;
(4)切片及展片:切片机5μm厚度切片,50℃水浴展片,捞片贴片于清洁载玻片上,60℃烘箱烤片过夜。切片完成后做好标记,保存待用。
2、H&E染色
(1)脱蜡和复水:切片二甲苯脱蜡两次(15分钟/次),分别于100%、95%、90%、80%、70%、50%酒精中各脱水5分钟,最后于蒸馏水中复水3分钟;
(2)苏木素染色:切片置于苏木素染液中染色15分钟,自来水冲洗3分钟,盐酸酒精分色10秒(70%酒精99ml+浓盐酸1ml);
(3)返蓝及脱水:自来水冲洗10分钟使其变为蓝色。将切片依次置于50%、70%、80%、90%酒精中各脱水5分钟;
(4)伊红复染:1%伊红染液染色2分钟,95%酒精和100%酒精中分别3分钟脱水分色至界限清楚;
(5)透明与封片:二甲苯透明3分钟后,中性树胶封片;
(6)封片后放入50℃烘箱烘干,光镜下观察肾组织病理学结构的变化。
实施例4油红染色对NAFLD肝脏组织病理学观察
(1)冰冻切片厚度6~10μm,10%福尔马林固定10min后水洗。
(2)切片入蒸馏水中稍冲洗。
(3)切片入60%的异丙醇内浸洗20~30s。
(4)切片入改良油红O染色液中(加盖),密闭染色10~15min。
(5)分色:入60%的异丙醇内稍洗以便去除染液。
(6)入蒸馏水中稍微清洗。
(7)Mayer苏木素染色液复染核1~2min。
(8)自来水漂洗10min促蓝。
(9)入蒸馏水中稍微清洗。
(10)用滤纸吸干周围水分,甘油明胶封片,显微镜下观察。
实施例5 NAFLD小鼠血清总胆固醇(TC)、甘油三酯(TG)试剂盒测定
收集动物全血,静置离心后取上清,按照要求测定TC含量。96孔板中每孔依次加入250μl工作液和2.5μl样本,轻轻震荡孔板,37℃恒温气浴10分钟,于510nm,酶标仪测定各个孔OD值,(绝对OD值=测定孔OD值-对照孔OD值),带入公式计算。
实施例6 NAFLD小鼠血清AST、ALT试剂盒测定
收集动物全血,静置离心后取上清,按照要求测定谷丙转氨酶或谷草转氨酶含量。96孔板中每孔依次加入20μl预温的基质液和5μl样本,轻轻震荡孔板,37℃恒温气浴30分钟。孵育后,立即加入20μl显色液。轻轻震荡孔板,37℃恒温气浴20分钟。加入200μl应用终止液。轻轻水平晃动96孔板,室温放置15分钟,于510nm,酶标仪测定各个孔OD值,(绝对OD值=测定孔OD值-对照孔OD值),带入标准曲线,计算得ALT或AST/GPT活力单位。
数据分析:
采用SPSS 16.0软件统计分析实验数据,两组比较采用t检验,多组比较采用单因素方差分析(one-way ANOVA),以平均值±标准误(Mean±SEM)表示,当P<0.05时,认为有统计学意义。
实验结果说明
图1为cPGES对照组(NC)小鼠和过表达组(OE)小鼠葡萄糖耐量实验(IPGTT)结果图。IPGTT实验结果用于指示对照小鼠以及过表达小鼠在葡萄糖耐受方面的差异,结果显示过表达cPGES之后的NAFLD小鼠的葡萄糖耐受性与对照组小鼠相比明显降低。
图2a-图2b为cPGES对照组(NC)小鼠和过表达组(OE)小鼠肝脏组织中cPGES表达水平图。免疫印迹常用于鉴定某种蛋白,并能对蛋白进行定性和半定量分析。结合化学发光检测,可以同时比较多个样品同种蛋白的表达量差异。结果显示过表达cPGES之后的NAFLD小鼠肝脏cPGES的蛋白表达水平升高。
图3a-图3b为cPGES对照组(NC)小鼠和过表达组(OE)小鼠肝脏组织形态图。HE染色用于观察组织形态改变情况,如图3a-图3b所示,cPGES过表达小鼠肝脏损伤明显加重,表现为肝脏空泡的明显增多。这提示cPGES的表达水平升高在NAFLD小鼠中明显加重了肝损伤。
图4a-图4b为cPGES对照组(NC)小鼠和过表达组(OE)小鼠肝脏油红染色结果图。油红染色可以观察肝脏脂肪的分布情况,具体颜色因脂质浓度而定。小鼠称体重,处死后解刨取肝脏,称肝脏重量。切小块肝组织在多聚甲醛中固定2-3天,之后分别在20%以及30%蔗糖溶液中沉糖,冰冻切片后染油红。如图4a-图4b所示,OE组的肝脏脂滴聚集明显增多,表明cPGES过表达加重了小鼠非酒精性脂肪肝病的发展。
图5a-图5b为cPGES对照组(NC)小鼠和过表达组(OE)小鼠血清TC、TG结果图。血液中的总胆固醇和甘油三酯水平是评估血脂代谢和心血管健康的重要指标。结果显示,过表达cPGES之后NAFLD小鼠血清TC、TG水平明显升高。
图6a-图6b为cPGES对照组(NC)小鼠和敲低组(KD)小鼠肝脏组织中cPGES表达水平图。处理方式同上,结果显示敲低cPGES之后的NAFLD小鼠肝脏cPGES的蛋白表达水平降低。
图7a-图7b为cPGES对照组(NC)小鼠和敲低组(KD)小鼠肝脏组织形态图。处理方式同上,HE染色用于观察肝脏形态和肝脏空泡占比,空白部分代表空泡,如图7a-图7b所示,KD组空泡占比明显下降,表明cPGES敲低改善非酒精性脂肪肝病。
图8a-图8b为cPGES对照组(NC)小鼠和敲低组(KD)小鼠肝脏油红染色结果图。处理方式同上,油红染色可以观察肝脏脂肪的分布情况,具体颜色因脂质浓度而定。如图8a-图8b所示,KD组小鼠肝组织不仅空泡减少而且脂质堆积出现了明显改善,这些结果表明cPGES敲低之后可以延缓NAFLD的进展。
图9a-图9b为cPGES对照组(NC)小鼠和敲低组(KD)小鼠血清AST、ALT结果图。AST、ALT水平升高是肝脏损伤的标志。如图9a-图9b所示,KD组小鼠血清学指标AST、ALT明显降低,这些结果表明cPGES敲低之后可以改善NAFLD小鼠肝脏损伤。
此外,本案发明人还参照前述实施例,以本说明书述及的其它原料、工艺操作、工艺条件进行了试验,并均获得了较为理想的结果。
应当理解,本发明的技术方案不限于上述具体实施案例的限制,凡是在不脱离本发明宗旨和权利要求所保护的范围情况下,根据本发明的技术方案做出的技术变形,均落于本发明的保护范围之内。
Claims (10)
1.胞质前列腺素E合酶作为靶点在开发或制备或筛选用于预防和/或治疗非酒精性脂肪肝病的药物中的应用。
2.根据权利要求1所述的应用,其特征在于:所述靶点过表达使得非酒精性脂肪肝病小鼠的葡萄糖耐受度降低;
和/或,所述靶点过表达使得非酒精性脂肪肝病小鼠的肝脏组织中胞质前列腺素E合酶的表达水平升高;
和/或,所述靶点过表达使得非酒精性脂肪肝病小鼠的肝脏脂质堆积;
和/或,所述靶点过表达使得非酒精性脂肪肝病小鼠的血清TC或血清TG水平升高。
3.根据权利要求1所述的应用,其特征在于:所述靶点敲低至少能够降低非酒精性脂肪肝病小鼠的肝脏组织中胞质前列腺素E合酶的表达水平;
和/或,所述靶点敲低至少能够抑制非酒精性脂肪肝病小鼠的肝脏脂质滴堆积;
和/或,所述靶点敲低至少能够降低非酒精性脂肪肝病小鼠的血清AST或血清ALT水平升高。
4.根据权利要求1所述的应用,其特征在于:所述非酒精性脂肪肝病包括胞质前列腺素E合酶过表达及高脂条件下诱导的非酒精性脂肪肝病。
5.胞质前列腺素E合酶或其编码基因的下调剂在制备用于预防和/或治疗非酒精性脂肪肝病的药物中的应用。
6.根据权利要求5所述的应用,其特征在于:所述下调剂选自特异性干扰胞质前列腺素E合酶的编码基因表达的干扰分子、特异性抑制胞质前列腺素E合酶或其编码基因的小分子化合物,或者特异性与胞质前列腺素E合酶结合的抗体或配体。
7.根据权利要求6所述的应用,其特征在于:所述干扰分子是以胞质前列腺素E合酶的编码基因或其转录本为抑制或沉默靶标的小干扰RNA、反义核酸、微小RNA、dsRNA,或能表达或形成所述小干扰RNA、反义核酸、微小RNA、dsRNA的构建物。
8.一种用于预防和/或治疗非酒精性脂肪肝病的药物组合物,其特征在于,包括:胞质前列腺素E合酶或其编码基因的下调剂,以及药学上可接受的载体。
9.根据权利要求8所述的药物组合物,其特征在于:所述下调剂选自特异性干扰胞质前列腺素E合酶的编码基因表达的干扰分子、特异性抑制胞质前列腺素E合酶或其编码基因的小分子化合物,或者特异性与胞质前列腺素E合酶结合的抗体或配体。
10.根据权利要求9所述的药物组合物,其特征在于:所述干扰分子是以胞质前列腺素E合酶的编码基因或其转录本为抑制或沉默靶标的小干扰RNA、反义核酸、微小RNA、dsRNA,或能表达或形成所述小干扰RNA、反义核酸、微小RNA、dsRNA的构建物。
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