CN111084769A - 天冬氨酸在制备防治非酒精性脂肪肝的药物中的应用 - Google Patents
天冬氨酸在制备防治非酒精性脂肪肝的药物中的应用 Download PDFInfo
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Abstract
本发明涉及天冬氨酸在制备防治非酒精性脂肪肝的药物中的应用。本发明首次探究了天冬氨酸参与NAFLD发生发展的重要生物学作用及其分子机制，揭示天冬氨酸代谢异常在NAFLD发展过程中的作用，为天冬氨酸干预治疗提供重要的理论基础；并通过体内外实验，首次明确了天冬氨酸的药性特征，建立评估其生物有效性和安全性的体系，为后续规范的药物临床前研究奠定基础。
Description
(一)技术领域
本发明涉及天冬氨酸在制备防治非酒精性脂肪肝的药物中的应用。
(二)背景技术
非酒精性脂肪性肝病(Nonalcoholic fatty liver disease，NAFLD)是 指除酒精和其他明确肝损伤因素所致，以弥漫性肝细胞大泡性脂肪变为主 要特征的临床病理综合征。近年有研究显示，我国成人NAFLD患病率已 达到20％左右，且呈逐年递增趋势；在美国等发达国家，成人NAFLD患 病率则更高达30％以上。NAFLD不仅是终末期肝病的主要病因，也能促 进肥胖、冠心病、糖尿病等代谢性疾病的发生发展[5]。虽然NAFLD现已 成为重点关注的肝脏疾病之一，其发病机制及诊治过程仍存在较多难点。
目前生活方式改善仍是NAFLD的基本治疗，以饮食干预和增加运动 为主，可降低血脂水平、改善胰岛素抵抗，但对进展期NAFLD，如非酒 精性脂肪性肝炎(Nonalcoholicsteatohepatitis，NASH)及肝硬化，仍需 辅助药物治疗。NAFLD目前已开发或正在开发的药物包括减轻脂质过氧 化的药物、胰岛素受体增敏剂、降血脂药、肝细胞保护剂、血管紧张素受 体拮抗剂、抗细胞因子抑制剂、肠道微生物调整剂等，然而改善脂变损伤、 纤维化等方面疗效有限。NAFLD的治疗尚缺乏特效干预手段，因此，迫 切需要对疾病新机制的探索及新药开发。
NAFLD的发病机制最为公认的仍是Day等人1998年提出的“二次打 击”学说：胰岛素抵抗引起的外周脂肪分解增加和高胰岛素血症，是导致 肝细胞脂肪变性的首要因素；脂肪变性的肝细胞对内、外源性损害因子的 敏感性增加，发生线粒体功能障碍，进一步导致炎症、肝细胞坏死和纤维 化构成第二次打击。其中，线粒体，作为细胞能量与代谢的关键细胞器， 在NAFLD发生发展中起关键作用。申请人前期研究发现(Gut,2018； 67(12):2169-2180；IF＝17.016)：线粒体代谢关键酶3-巯基丙酮酸转硫酶 (3-mercaptopyruvatesulfurtransferase，MPST)在NAFLD中高表达，部 分抑制MPST可以显著减轻NAFLD，揭示了NAFLD发生发展的新机制， 并为NAFLD药物开发提供新靶点。
MPST是肝细胞内线粒体硫化氢(Hydrogen sulfide，H2S)代谢途径 关键酶，可参与包括抗氧化、H2S生成调控及超氧化物合成，基因缺陷可 导致遗传学巯基乳酸盐二硫化半胱氨酸尿症，也与心血管疾病、神经系统 疾病、肿瘤、糖尿病、细菌感等疾病发生有关[15,16]。申请人前期研究发 现：在肝脏脂变模型中，MPST表达升高；MPST敲低后，胞内H2S水平 增加，可保护肝脏脂变损伤；MPST通过CSE直接相互作用调控H2S生 成；MPST直接与脂代谢相关基因固醇调节元件结合蛋白1c(Sterol Regulatory Element Binding Protein1c，SREBP1c)结合调节其活性，通过 下游脂代谢相关酶类(Fatty Acid Synthase，FAS)和(Acetyl-CoA Carboxylase，ACC)等参与肝脏脂变损伤。上述研究提示，抑制MPST 可保护NAFLD，因此，MPST抑制剂开发可作为NAFLD新药研发方向。
(三)发明内容
本发明目的是提供天冬氨酸在制备防治非酒精性脂肪肝的药物中的 应用。
本发明采用的技术方案是：
天冬氨酸在制备防治非酒精性脂肪肝的药物中的应用。
MPST含3个半胱氨酸残基Cys247、Cys154、Cys263，为经典的酶 活调控位点，可感知细胞氧化还原状态，可被内源性过氧化氢、四硫酸盐 等抑制，也可通过外源性DTT、DTNB、DNB-CI等药物直接激活。天冬 氨酸是机体非必需氨基酸，参与氨基酸、嘌呤合成及尿素循环等生物过程， 近年来报道天冬氨酸可竞争性抑制半胱氨酸氨基转移酶(cysteineaminotransferase，CAT)，抑制MPST活性。申请人前期研究表明：天冬 氨酸在NAFLD患者及NAFLD的小鼠的血清中含量显著下降；在NAFLD 小鼠模型中，通过饮水及饲料中添加天冬氨酸干预，可显著改善NAFLD， 因此天冬氨酸可用于制备防治非酒精性脂肪肝的药物。
本发明首次探究了天冬氨酸参与NAFLD发生发展的重要生物学作 用及其分子机制，揭示天冬氨酸代谢异常在NAFLD发展过程中的作用， 为天冬氨酸干预治疗提供重要的理论基础；并通过体内外实验，首次明确 了天冬氨酸的药性特征，建立评估其生物有效性和安全性的体系，为后续 规范的药物临床前研究奠定了基础。
具体的，所述天冬氨酸为L-天冬氨酸。
优选的，所述天冬氨酸用于制备改善肝脏脂质沉积的药物。
本发明的有益效果主要体现在：本发明提供了天冬氨酸在制备防治非 酒精性脂肪肝的药物中的新应用，为新药筛选提供了基础。
(四)附图说明
图1为高脂饮食喂养C57BL/6小鼠构建NAFLD细胞模型镜检肝组织变 化结果；A为正常对照(常规饲料喂养)，B为脂肪肝(高脂饮食喂养)。
图2为NAFLD患者血清、动物模型及细胞模型中L-天冬氨酸含量检测 结果；A：健康人群及NAFLD患者血清ASP含量；B：正常小鼠及HFD 造模16周小鼠及饮水中给与不同浓度ASP小鼠血清ASP含量；C：正常 小鼠及HFD造模16周小鼠及饮水中给与不同浓度ASP小鼠肝组织中 ASP含量；D和E：7701细胞内(D)及L02细胞内(E)ASP含量。
图3为C57小鼠HFD造模16周，天冬氨酸干预对肝脏脂变影响；A： HFD造模16周及造模同时给与10mM ASP肝脏大体情况；B：HFD造模 16周及造模同时给与10mM ASP肝脏病理情况；C：HFD造模16周及造 模同时给与10mM ASP肝脏油红情况；D：HFD造模16周及造模同时给与5mM ASP及10mM ASP肝脏TG水平；E：体外实验给与5mM ASP 及1mM ASP的原代肝细胞内TG水平；F：体外实验给与5mM ASP及 1mM ASP的7701细胞内TG水平。
图4为有效剂量下ASP对小鼠多器官功能影响；A和B：10mM ASP下 ASP对肝功能的影响；C：10mM ASP对血清BUN影响；D：10mM ASP 对血清creatinine的影响；E及F：10mM ASP对心功能影响，E示血清 CK，F示血清LDH。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围 并不仅限于此：
实施例1：
NAFLD小鼠模型构建：NAFLD小鼠模型建立及鉴定：8-12周龄雄 性C57BL/6小鼠高脂饮食(HFD:含60％脂肪，购自Research Diets公司， 货号D12492)或常规饲料(SCD)喂养12周，采用全自动生化检测仪检 测血清ALT、AST、TG水平，采用上述油红-苏木素染色、HE染色、 Masson染色后镜检肝组织脂肪变性等病理学变化，结果见图1。检测结 果与常规饲养小鼠比较后显示成功构建了NAFLD小鼠模型。
天冬氨酸测定：将天冬氨酸检测试剂盒(sigma)-20℃取出后室温放 置半小时。
检测前处理：收集健康人群及NAFLD患者全血标本，3000转离心 10min，取100ul上清，加入2ul Serum Clean Up Mix，室温孵育30min后 转移至10kDa MWCO离心管，12000转离心10min，收集下层上清备用； 取动物组织，精确称量，加入10mg/ul assay buffer，匀浆后12000转离心 10min，取上清备用；收集细胞，加入100ul assay buffer，超声裂解后12000 转离心10min后取上清待测；按照说明书稀释标准品，将前处理好的样 品及标准品各50ul加入96孔板，每空加入44ul assay buffer，2ul Enzyme Mix，2ul Conversion Mix，2ul Probe后混匀室温避光孵育30min，570nm 检测吸光度，根据标准曲线计算天冬氨酸含量。
结果见图2，显示L-天冬氨酸在NAFLD患者血清，动物模型及细胞 模型中含量下降，外源性ASP补充明显提高小鼠肝脏和肝脏细胞内ASP 含量。
天冬氨酸干预实验：在体内实验中8周龄雄鼠32只，分为SCD饮食； SCD+10mM天冬氨酸；HFD饮食；HFD+10mM天冬氨酸，共4组，按 每组不同浓度计算所需天冬氨酸，精确称取L-天冬氨酸(sigma)，溶于 饮水中，涡旋30min促溶。隔天更换饮水，每周称量体重及进食量。在 细胞实验：原代肝细胞或7701细胞铺板后贴壁过夜，称取不同浓度天冬 氨酸溶于培养基或FFA中，37度水浴30min促溶。更换普通培养基为含 天冬氨酸培养基，培养24h后检测细胞内ASP，TG。
结果见图3，在体内实验中，和HFD喂养相比，饮水中添加ASP明 显改善肝脏体积大小(3A)，病理和油红证实肝脏脂滴含量明显减少(3B 及3C)，肝脏TG水平也证实以上现象(3D)，在体外实验中，ASP添加 也明显改善FFA诱导的原代肝细胞(3E)及7701(3F)细胞内TG水平， 以上结果提示天冬氨酸干预明显改善肝脏脂质沉积。
安全性早期评价：
将40只健康6～8周龄C57BL/6小鼠随机分为4组，每组10只(雌 雄各半)，通过前述天冬氨酸干预方法，取有效剂量10mM进行干预，共 8周。每日观察并记录小鼠一般情况(精神、摄食、活动、体重、毛色等)， 8周后收集血清检测ALT，AST，creatinine，BUN，CK，LDH，收集小 鼠心、肝、肾、肺、脂肪、肠道标本进行组织病理学分析，评估细胞形态、 凋亡、组织损伤等。
结果见图4，显示L-天冬氨酸在改善肝脏脂质沉积时的浓度对心功 能，肝功能，及肾功能无明显损害。
Claims (4)
1.天冬氨酸在制备预防非酒精性脂肪肝的药物中的应用。
2.天冬氨酸在制备治疗非酒精性脂肪肝的药物中的应用。
3.如权利要求1或2所述的应用，其特征在于所述天冬氨酸为L-天冬氨酸。
4.如权利要求1或2所述的应用，其特征在于所述天冬氨酸用于制备改善肝脏脂质沉积的药物。
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| CN111621561A (zh) * | 2020-06-15 | 2020-09-04 | 浙江大学 | Olfm4在非酒精性脂肪肝（nafld）中的应用 |
| CN115120695A (zh) * | 2022-08-31 | 2022-09-30 | 健码制药（广东）有限公司 | 一种甘草益生菌复合制剂clpp-9及其在预防或治疗非酒精性脂肪性肝病的应用 |
| WO2023083291A1 (zh) * | 2021-11-12 | 2023-05-19 | 深圳利沃生物医药科技有限公司 | 天冬氨酸的衍生物及其在肝纤维化、非酒精性肝炎等代谢等疾病的治疗的应用 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111621561A (zh) * | 2020-06-15 | 2020-09-04 | 浙江大学 | Olfm4在非酒精性脂肪肝（nafld）中的应用 |
| WO2023083291A1 (zh) * | 2021-11-12 | 2023-05-19 | 深圳利沃生物医药科技有限公司 | 天冬氨酸的衍生物及其在肝纤维化、非酒精性肝炎等代谢等疾病的治疗的应用 |
| CN115120695A (zh) * | 2022-08-31 | 2022-09-30 | 健码制药（广东）有限公司 | 一种甘草益生菌复合制剂clpp-9及其在预防或治疗非酒精性脂肪性肝病的应用 |
| CN115120695B (zh) * | 2022-08-31 | 2022-12-06 | 健码制药（广东）有限公司 | 一种甘草益生菌复合制剂及其在预防或治疗非酒精性脂肪性肝病的应用 |
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