CN113209118A - 一种治疗非酒精性脂肪性肝炎的药物 - Google Patents
一种治疗非酒精性脂肪性肝炎的药物 Download PDFInfo
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Abstract
本发明属于医药领域，提供了一种用于治疗非酒精性脂肪性肝炎(NASH)药物。本发明发现，化合物金线莲苷(Kinsenoside(3‑(R)‑3‑β‑D‑Glucopyranosyloxybutanolide)对采用MCD饲料(蛋氨酸‑胆碱缺乏饲料)诱导的小鼠非酒精性脂肪性肝炎(NASH)具有较好的治疗作用，能够有效减少肝脏脂肪囤积、改善肝脏损伤、减少炎症浸润和氧化应激损伤、并降低纤维化标志基因和蛋白的表达从而阻止NASH向肝纤维化的发展，揭示金线莲苷能作为治疗非酒精性脂肪性肝炎的药物用于治疗非酒精性脂肪性肝炎(NASH)。
Description
技术领域
本发明属于医药技术领域，涉及治疗肝脏疾病的药物，具体涉及治疗非酒精性脂肪性肝炎(NASH)的药物，即一种糖苷类化合物在制备用于治疗NASH药物中的应用。
背景技术
随着我国经济的发展,人民生活水平不断提高,人们的生活方式发生了明显的改变,非酒精性脂肪性肝病的发病率持续增加,目前该疾病已经成为我国最常见的慢性肝病之一。非酒精性脂肪性肝病主要是指除明确肝损伤以及酒精等因素外导致的脂肪在肝细胞内部过度沉积,疾病谱包括非酒精性单纯性脂肪肝、非酒精性脂肪性肝炎及其相关肝硬化和肝细胞癌,患者一旦出现该类疾病,极易引发肝酶异常。诱发非酒精性脂肪性肝病的原因较为复杂,加强诊断和防治刻不容缓。(参考文献：王凤谨，非酒精性脂肪性肝病诊断和防治进展[J].中外女性健康研究)。
非酒精性脂肪肝病(NAFLD)是一种进展性肝疾病,其发展始于脂肪肝,经脂肪性肝炎(NASH)而后演变成肝纤维化和肝硬化。NAFLD的病理分子机制尚不完全清楚,也无有效治疗方法。目前,认为肝过度摄入脂肪和氧化应激在NAFLD的病理发展过程中有十分重要的作用。MCD引起的脂肪性肝炎模型最初由Shinozuka提出,在该模型中脂肪性肝炎发展速度较快,4～6周左右可见明显的脂肪性肝炎病变,8周可见肝纤维化,其引起的病理病变与人类的NASH类似。MCD是指在喂养饲料中选择性地不含有蛋氨酸及胆碱，其作用机理在于减少了脂蛋白的合成，尤其是极低密度脂蛋白，造成了甘油三酯在肝脏细胞的沉积，并且损伤了抗氧化屏障机制导致了氧化应激加剧，在非酒精性脂肪性肝纤维化发生和发展的多个环节都产生作用。MCD诱导的小鼠肝纤维化模型造模方法简单，模型建成时间短，适合用于非酒精性脂肪性肝炎发病机制及药物干预等方面的研究。目前临床对于NASH的研究仍缺乏确定有效的药物，现有的水飞蓟素等药物仅能够延缓疾病的发展，对于逆转疾病则无明显效果。因此，探索研究开发一种治疗NASH的有效药物，是该领域长期以来的重要课题。
天然产物是药物最常见的来源，据统计，从上世纪八十年代年至今，每年30-40％的上市药物直接或间接来源于天然产物，其中2010年，天然产物相关来源的药物比例高达50％。植物次生代谢产物的种类极为丰富，随着近年对其研究的不断增多，越来越多活性强的化合物被发现，为新药的研发奠定了一定的基础。本发明旨在从天然产物中寻找治疗NASH的有效药物。
发明内容
本发明的提供了一种治疗NASH的药物。实现本发明的技术方案是：本发明提供治疗NASH的药物是金线莲苷(Kinsenoside(3-(R)-3-β-D-Glucopyranosyloxybutanolide))。金线莲苷(KD)是从金线莲中分离出来的具有生物活性的小分子化合物，金线莲是有“药王”之称的传统草本植物，广泛分布于印度、喜马拉雅山、东南亚、夏威夷等热带地区。抗高血糖、抗高脂血症、抗骨质疏松和抗疲劳等是其重要的药理作用，也有文献表明KD有免疫抑制、抗高血糖和抗高脂血症作用，可缓解急性炎症，但保肝作用最为显著(参考文献：何春年，王春兰，郭顺星，等.兰科开唇兰属植物的化学成分和药理活性的研究[J].中国药学杂志)。对于金线莲苷的保肝护肝活性研究，目前主要集中在四氯化碳诱导的小鼠急、慢性肝损伤模型以及ConA诱导的自身免疫性肝炎模型等。但对于金线莲苷的应用仅见研究性报道，该化合物尚无正式药物上市，采用MCD饲料(蛋氨酸-胆碱缺乏饲料)诱导小鼠的NASH模型以探究金线莲苷对非酒精性脂肪性肝炎的作用的研究也尚未开展，发明人针对这一问题，对金线莲苷治疗MCD饲料所致NASH伴肝纤维化模型的作用进行了探讨，并取得了较显著的成果。
金线莲苷属于糖苷类化合物，金线莲苷的结构式如式1所示：
本发明研究使用的金线莲苷购买自上海同田生物技术股份有限公司，纯度>98.0％。HPLC测定KD纯度色谱图见图4。
以下为核磁共振谱图鉴定KD数据。
本发明人通过对化合物金线莲苷的治疗NASH活性进行评价，发现该化合物能够有效阻止NASH的发生发展，在体内产生一定的保护肝损伤、改善肝功能、降低肝脏脂肪蓄积、减少炎症浸润和氧化应激损伤、降低纤维化标志基因表达阻止NASH向肝纤维化的进展等活性，可以作为治疗NASH药物开发的先导化合物。
附图说明
图1：运用油红O染色、Image J软件分析脂滴面积以及血清生化试剂盒(AST、ALT、TG、TC)来研究KD对NASH小鼠肝脏脂肪变性的保护作用。在图1中：
A.肝脏油红O染色(200X)。结果显示，造模2周后即形成单纯性脂肪肝(NAFL)，4周以后脂肪变性持续加重，而KD高剂量组(40mg/kg KD)、中剂量组(20mg/kg KD)和低剂量组(10mg/kg KD)均可显著减少肝脏脂肪蓄积。
B.用Image J软件分析8w时各组切片的脂滴面积，发现KD各剂量组均能显著减少肝内脂肪堆积，KD高剂量组的效果甚至优于阳性药组。
C.肝指数。肝指数为小鼠的肝脏与自身体重之比，与正常组小鼠相比，模型组小鼠肝指数升高，提示肝内脂肪蓄积，而KD各剂量组均能减少肝内脂肪，使肝指数趋于正常。
D、E.肝功能指标AST、ALT。造模后，小鼠血清转氨酶水平显著升高，KD各剂量组均能不同程度地降低AST、ALT，改善肝功能，且结果具有显著性差异。
F、G、H、I.用生化试剂盒检测小鼠血清和肝匀浆中的脂肪TG、TC含量。结果显示KD各剂量组均能显著降低MCD所致NASH小鼠肝脏中的脂肪含量，同时使血脂含量略有回升。
图2：用HE染色、CBA流式试剂盒检测血清中的炎性因子含量来探究KD对NASH小鼠炎症的影响；用生化试剂盒(SOD、MDA)来测定各组小鼠中氧化应激水平；用RT-qPCR、蛋白免疫印迹实验)(Western Blot)以及免疫组化染色法来研究KD对细胞凋亡的影响。在图2中：
A.肝脏HE染色(200X)。HE染色结果显示，造模2周后即形成单纯性脂肪肝(NAFL)，4周开始有炎性病灶产生，6周形成脂肪性肝炎(NASH)；而KD各剂量组能抑制肝脏的炎性浸润。
B、C、D、E、F、G.CBA流式试剂盒检测小鼠血清中炎性因子含量。与正常对照组相比，模型组促炎因子IL-6、IL-12p70、MCP-1、TNF-α、IFN-γ的含量显著升高，而抑炎因子IL-10含量有所下降，KD各剂量组可以降低促炎因子的血清含量同时升高抑炎因子IL-10从而减轻炎症反应。
H、I.生化试剂盒检测肝脏中超氧化物歧化酶(SOD)及氧化产物丙二醛(MDA)的含量。造模后，模型小鼠肝脏中SOD含量下降，同时MDA显著上升，提示NASH小鼠氧化应激加剧，而给予KD治疗后，肝脏中SOD含量上升，氧化产物MDA减少，说明KD能改善NASH小鼠的氧化应激反应。
J、K、L.用RT-qPCR检测肝脏中凋亡相关基因的表达。结果显示，与正常组相比，模型组促凋亡基因Bax显著升高，抗凋亡基因Bcl-2表达下调，而凋亡标志基因Casp3也显著升高；而KD高剂量组能显著升高抗凋亡基因Bcl-2的表达，下调促凋亡基因Bax的表达，使得凋亡标志基因Casp3表达降低，发挥抗凋亡作用。
M.用Western Blot检测肝脏中凋亡相关蛋白的表达。模型组中促凋亡蛋白BAX含量升高，而抗凋亡蛋白Bcl-xl表达减少；给予KD治疗后，抗凋亡蛋白Bcl-xl表达升高，促凋亡蛋白BAX收到抑制，提示KD具有抗凋亡作用。
N.免疫组化染色法来研究肝脏中凋亡标志蛋白Cleaved Caspase3(CC3)的表达。与正常组相比，模型组CC3表达增多，提示模型小鼠肝细胞凋亡严重，KD个剂量组均能不同程度地减少肝细胞凋亡，从而降低肝脏CC3蛋白的表达。
图3：用Masson染色、ELISA试剂盒(HA、LN、C-Ⅳ、PCⅢ)、RT-qPCR、超声波瞬时剪切技术、Western Blot以及免疫组化染色法来研究KD对肝纤维化的影响。在图3中：
A.肝脏Masson染色(200X)。Masson染色结果表明，MCD喂养小鼠到8w时开始由NASH进展为肝纤维化，模型组肝脏内被染成蓝色的纤维增多，但KD各剂量组均能不同程度减少纤维生成。
B.用Image J软件分析8w时各组切片的纤维面积，结果显示模型组纤维面积显著升高，给予KD治疗后，肝脏纤维含量减少，KD的效果甚至优于阳性药组。
C、D、E、F.用ELISA试剂盒测定血清“肝纤维化四项”的含量。结果显示，模型小鼠血清中HA、LN、C-Ⅳ、PCⅢ四项指标均显著升高，KD各剂量组能显著降低HA、LN的含量，对肝纤维化起到一定抑制作用。
G.qPCR检测肝组织中纤维化标志基因表达情况。结果也显示，KD可有效抑制小鼠肝脏α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、一型胶原(collagen-I,Col I)、基质金属蛋白酶抑制剂-1(tissue inhibitor of metalloproteinase-1,TIMP-1)、转化生长因子(transforming growth factor-β,TGF-β)的表达，上调基质金属蛋白酶-13(matrixmetalloproteinase-13,MMP-13)的表达。
H.用超声波瞬时剪切技术来测定小鼠肝脏的硬度值。剪切速度越大，说明小鼠肝脏越硬，在图中表示越偏向红色。
I.用Image J软件量化H图中各组肝脏的硬度值。与正常组相比，模型组肝脏硬度增大，提示纤维化增生严重，而KD各剂量组的肝脏硬度值都有所下降，说明KD能一定程度抑制NASH小鼠向肝纤维化进展。
J、K Western Blot和免疫组化实验测定肝纤维化标志蛋白α-SMA的表达。结果显示，KD各剂量组均能减少α-SMA的表达，抑制肝纤维化。
图4：HPLC测定KD纯度色谱图。
具体实施方式
实施例1
金线莲苷(kinsenoside；KD)在治疗NASH中的作用。
本实验购入6-8周龄的SPF级雄性C57鼠150只，饲养于华中科技大学同济医学院SPF级实验动物中心，给予标准饮食和饮水，房间温度控制在23±2℃，适应喂养7天后开始灌胃给药。实验中涉及小鼠的实验方案经华中科技大学同济医学院动物实验伦理委员会批准。小鼠随机分为空白组(control group)、模型组(model group)、KD低剂量组(40mg/kgKD)、KD中剂量组(20mg/kg KD)、KD高剂量组(10mg/kg KD)、阳性药水飞蓟素组(100mg/kg)和阳性药扶正化瘀组(1.5g/kg)。其中模型组和各给药组给予MCD(蛋氨酸-胆碱缺乏)饲料喂养，正常对照组以正常饲料喂养。各小组均采用灌胃的方式给药，正常组和模型组给予等量三蒸水，每天1次，持续给药8w,每三天称一次小鼠体重，观察各组小鼠的毛色，状态，食量饮水及大小便情况，并记录各组小鼠的死亡情况。在给药的第2w、4w、6w末分别随机选取3只小鼠处死取材观察成模情况，第8周末最后一次给药后将所有小鼠禁食不禁水12h，取材测定各项指标。
造模2周后即形成单纯性脂肪肝(NAFL)，4周开始有炎性病灶产生，6周形成脂肪性肝炎(NASH)，8周逐渐进展为肝纤维化(图1A、2A、3A)。结果显示，不同剂量的KD均能显著降低血清ALT、AST水平、减少肝脏系数，改善肝功能(图1C、D、E)；肝脏油红O染色显示，在高剂量组(40mg/kg KD)、中剂量组(20mg/kg KD)和低剂量组(10mg/kg KD)中，KD可显著减轻肝脏脂肪蓄积(图1A、B)；图1F、G、H、I显示KD各剂量组均能显著降低MCD所致NASH小鼠肝脏中的脂肪含量，同时使血脂含量略有回升。HE染色结果显示，模型组小鼠从4周开始出现炎性病灶，6周以后进展成为NASH(图2A)；与正常对照组相比，模型组促炎因子IL-6、IL-12p70、MCP-1、TNF-α、IFN-γ的含量显著升高，而抑炎因子IL-10含量有所下降，KD各剂量组可以降低促炎因子的血清含量同时升高抑炎因子IL-10从而减轻炎症反应(图2B、C、D、E、F、G)；模型组脂质过氧化反应产物丙二醛MDA表达上升，超氧化物歧化酶(SOD)活性下降，给予KD治疗可以逆转这些氧化应激损伤(图2H、I)；此外，KD还能下调促凋亡基因和蛋白表达，升高抗凋亡基因的表达从而减少肝细胞凋亡(图2J、K、L、M、N)。Masson染色显示，MCD饲料造模8周时，模型小鼠开始进展为肝纤维化，而KD各剂量组均对纤维形成有抑制作用(图3A、B)；采用实时剪切波弹性成像(SWE)超声技术检测小鼠肝纤维化发展情况以及KD的治疗潜能，SWE的剪切波速度测量结果显示，模型组小鼠肝脏的回声速度显著增大，提示肝组织硬度大，肝脏纤维化病变变严重，而KD可显著降低剪切波速度，缓解肝纤维化(图3H、I)；用ELISA试剂盒检测小鼠血清中“肝纤维化四项指标(HA、LN、Col-Ⅳ和PCⅢ)”的含量，发现模型组小鼠的四项指标较正常组均显著升高，KD各剂量组均能不同程度地降低HA和LN含量(3C、D、E、F)；qPCR检测肝组织中纤维化标志基因表达情况，结果也显示，KD可有效抑制小鼠肝脏α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、一型胶原(collagen-I,Col I)、基质金属蛋白酶抑制剂-1(tissue inhibitor of metalloproteinase-1,TIMP-1)、转化生长因子(transforming growth factor-β,TGF-β)的表达，上调基质金属蛋白酶-13(matrixmetalloproteinase-13,MMP-13)的表达(图3G)；蛋白免疫印迹实验和免疫组化实验显示KD各剂量组均能减少纤维化标志蛋白——α-SMA的表达(图3J、K)。
综上，KD的治疗作用呈现出一定剂量依赖性，作用效果与阳性药水飞蓟素、扶正化瘀相当，在某些指标中效果优于阳性药。与此同时，阴性对照组给予健康小鼠KD，不影响其ALT、AST和其他正常功能。
实验结论：金线莲苷对MCD诱导的NASH具有较好的治疗作用，能够有效减少肝脏脂肪囤积、改善肝脏损伤、减少炎症浸润改善肝功能和氧化应激损伤、并降低纤维化标志基因表达阻止NASH向肝纤维化的发展。
Claims (3)
1.金线莲苷(Kinsenoside(3-(R)-3-β-D-Glucopyranosyloxybutanolide)在制备用于治疗非酒精性脂肪性肝炎(NASH)药物中的应用。
2.一种用于治疗非酒精性脂肪性肝炎(NASH)的药物制剂，其特征在于，含有效量的金线莲苷(Kinsenoside(3-(R)-3-β-D-Glucopyranosyloxybutanolide)和制药学上可接受的添加剂、载体或/和赋型剂。
3.权利要求1或2中所述的金线莲苷(Kinsenoside(3-(R)-3-β-D-Glucopyranosyloxybutanolide)为以下式1所示的化合物。
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