CN115160404A - 多肽化合物在制备用于预防或治疗非酒精性脂肪肝病药物中的应用 - Google Patents
多肽化合物在制备用于预防或治疗非酒精性脂肪肝病药物中的应用 Download PDFInfo
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Abstract
本发明涉及一类多肽化合物在制备用于预防或治疗非酒精性脂肪肝病药物中的应用,该多类肽化合物具有高酶解稳定性、高生物活性、无不良反应等特点,能够改善治疗非酒精性肝病包括:脂肪肝、肝脏损伤及炎症和纤维化程度。该类多肽化合物可用于预防或治疗非酒精性脂肪肝病以及诱发相关炎症和纤维化等疾病。
Description
技术领域
本发明属于生物化学技术领域,具体地,涉及一类激动剂多肽化合物。本发明还涉及上述激动剂多肽化合物对非酒精性脂肪肝病的预防和/或治疗用途。
背景技术
非酒精性脂肪肝病(Nonalcoholic fatty liver disease,NAFLD)已被公认为世界范围内引发慢性肝脏疾病的主要病因。基于最新的数据调查结果显示,全世界有25%的成年人会受到NAFLD的影响,而在孩童中,NAFLD的普及度也已达到10%。NAFLD是因肝脏功能异常而导致的一系列疾病,包括单纯的脂肪变性,以及由其演变的非酒精性脂肪肝炎(Non-alcoholic steatohepatitis,NASH)、肝纤维化、肝硬化,甚至是肝癌。
尽管对于NAFLD的发病机制还存在很多不确定性,但“二次打击假说”是目前公认的用于解释NAFLD发展的主要理论。第一次打击会造成脂肪变性,其与胰岛素抵抗(insulinresistance,IR)密切相关,伴随着脂质生成及脂肪酸输出受损。第二次打击涉及到活性氧(reactive oxygen species,ROS)的输出,从而造成系统性氧化应激,进一步导致炎症和细胞毒性,最终造成NASH和肝纤维化。SchohrayaSpahis小组提到了第三次打击假说,表明过度的氧化应激产生还会导致肝细胞的死亡,成熟肝细胞复制减少,造成肝硬化甚至是肝癌。总之,在肝脏中的脂肪累积(第一次打击)降低了肝脏对氧化应激的抵抗能力(第二次打击),从而引发炎症,内质网(endoplasmic reticulum,ER)应激,线粒体损伤以及合成内源性抗氧化剂的能力减弱等。因此,在由多种因子导致的NAFLD疾病中,氧化应激在促进NAFLD肝脏损伤中发挥着很重要的作用。综上所述,抗氧化作用可以作为NAFLD的一种有效治疗手段。
目前仍然缺乏有效的治疗NAFLD/NASH的药物,PPAR-γ类胰岛素增敏剂、奥贝胆酸等法尼酯衍生物X受体(FXR)激动剂作为新型的在研药物,其长期使用安全性以及治疗有效性均有待进一步证明(Armstrong MJ,Gaunt P,Aithal GP,et al.Lancet,2015.doi:10.1016/S0140-6736(15)00803-X.)。
Feiran Xu小组从菜籽中分离出菜籽蛋白,并进一步获取了其主要活性成分,该活性成分的氨基酸序列为YWDHNNPQIR,其在人克隆结肠腺癌细胞中展示了很好的抗氧化作用,之前已对该活性成分做进一步研究,发现其在非酒精性脂肪肝病和肝纤维化疾病方面,展示了很好的肝保护作用。
本发明旨在对该活性片段进行深入剖析,分析其活性位点,并进行构效关系研究,以期获得活性更好,半衰期更长的多肽化合物。
发明内容
本发明的目的在于提供一种治疗非酒精性肝脏疾病的多肽化合物,从而为非酒精性脂肪肝病的治疗提供一种新的药物。
本案发明人建立了细胞模型和动物模型用以模拟人的非酒精性脂肪肝病,对菜籽蛋白的活性成分以及类似物进行体外和体内药效评估,筛选出了活性更好的多肽化合物,研究结果表明该多肽化合物在预防或治疗非酒精性脂肪肝病方面具有显著的效果。
本发明中涉及的多肽化合物含有以下氨基酸序列表示的母体肽:
R1-Xaa1-Xaa2–Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-COR2
其中,R1=-NH2或-Ac;R2=-NH2或-OH;
Xaa1=Tyr,Ala或D-Tyr;
Xaa2=Trp或Ala;
Xaa3=Asp,Ala或Glu;
Xaa4=His或Ala;
Xaa5=Asn,Ala或Gln;
Xaa6=Asn,Ala,Arg,Gln或Asp;
Xaa7=Pro或Ala;
Xaa8=Gln,Ala或Asn;
Xaa9=Ile,Ala或Leu;
Xaa10=Arg或Ala。
当所述母体肽的氨基酸序列为R1-Tyr-Trp-Asp-His-Asn-Asn-Pro-Gln-Ile-Arg-COR2时,R1≠-NH2。
上述多肽化合物和/或编码所述多肽化合物的核酸分子在制备治疗NAFLD的产品中的应用以及以上述多肽化合物和/或编码所述多肽化合物的核酸分子为活性成分的治疗NAFLD的产品均属于本发明的保护范围。此外,多肽化合物也可与亲脂性的取代基、蛋白和抗体中的至少一种衍生物和/或结构相连,相连形成多肽化合物同样属于本发明的保护范围。
优选地,本发明的母体肽的氨基酸序列选自SEQ ID NO:2,SEQ ID NO:3,SEQ IDNO:4,SEQ ID NO:5,SEQ ID NO:6,SEQ ID NO:7,SEQ ID NO:8,SEQ ID NO:9,SEQ ID NO:10,SEQ ID NO:11,SEQ ID NO:12,SEQ ID NO:13,SEQ ID NO:14,SEQ ID NO:15,SEQ IDNO:16,SEQ ID NO:17,SEQ ID NO:18,SEQ ID NO:19,SEQ ID NO:20,SEQ ID NO:21和SEQID NO:22中的一种。
更优选地,母体肽的氨基酸序列选自SEQ ID NO:14,SEQ ID NO:15,SEQ ID NO:16,SEQ ID NO:17,SEQ ID NO:18,SEQ ID NO:19,SEQ ID NO:20,SEQ ID NO:21和SEQ IDNO:22中的一种。
本发明还提供含有本发明的多肽化合物的药物组合物,以所述多肽化合物作为活性成分添加药学上可接受的载体和/或辅料制成药物组合物。
本发明的多肽化合物对非酒精性脂肪性肝炎、肝纤维化疾病具有改善和治疗作用。本发明的多肽化合物可用于直接或间接治疗由非酒精性脂肪肝炎和肝纤维化所引起的或者以其为特征的病症。所述NAFLD包括单纯的脂肪变性、非酒精性脂肪肝炎、肝纤维化、肝硬化和肝癌。所述改善和治疗NAFLD疾病可体现为脂肪变性的肝细胞数目减少、炎症减轻、细胞坏死和纤维化区域变小。
本领域技术人员可以理解,本发明的药物组合物适用于各种给药方式,例如口服给药、经皮给药、静脉给药、肌肉内给药、局部给药、经鼻给药等。根据所采用的给药方式,可将本发明的药物组合物制成各种合适的剂型,其中包含至少一种有效量的本发明的多肽化合物和至少一种药学上可接受的药用载体。
适当剂型的实例为片剂、胶囊、糖衣片剂、粒剂、口服溶液和糖浆、用于皮肤表面的油膏和药贴、气雾剂、鼻喷剂、以及可用于注射的无菌溶液。含有本发明多肽化合物的药物组合物可以制成溶液或者冻干粉以用于胃肠外给药,在使用前可加入适当溶剂或其他可药用的载体将粉末重新配制,液体配方一般是缓冲液、等渗溶液和水溶液。
本发明多肽化合物在药物组合物中的用量可以在较大范围内变动,本领域技术人员可以根据一些客观的因素如疾病的种类、病情严重程度、患者体重、剂型、给药途径等因素很容易地加以确定。
与现有技术相比,本发明的优点在于:
1)本发明的多肽化合物具有更好的改善和治疗非酒精性脂肪肝病的生物学活性;
2)与小分子化合物相比,本发明的多肽化合物具有更低毒性,安全窗口更大,用量更小;
3)与大分子蛋白相比,本发明的多肽化合物制备成本更经济,可推广性更高。
在具体的实施方案中,本发明涉及的多肽化合物具有以下氨基酸序列:
化合物1(涉及SEQ ID NO:1):
NH2-Tyr-Trp-Asp-His-Asn-Asn-Pro-Gln-Ile-Arg-CONH2
NH2-YWDHNNPQIR-CONH2
化合物2(涉及SEQ ID NO:2):
Ac-Tyr-Trp-Asp-His-Asn-Asn-Pro-Gln-Ile-Arg-CONH2
Ac-YWDHNNPQIR-CONH2
化合物3(涉及SEQ ID NO:3):
NH2-Ala-Trp-Asp-His-Asn-Asn-Pro-Gln-Ile-Arg-CONH2
NH2-AWDHNNPQIR-CONH2
化合物4(涉及SEQ ID NO:4):
NH2-Tyr-Ala-Asp-His-Asn-Asn-Pro-Gln-Ile-Arg-CONH2
NH2-YADHNNPQIR-CONH2
化合物5(涉及SEQ ID NO:5):
NH2-Tyr-Trp-Ala-His-Asn-Asn-Pro-Gln-Ile-Arg-CONH2
NH2-YWAHNNPQIR-CONH2
化合物6(涉及SEQ ID NO:6):
NH2-Tyr-Trp-Asp-Ala-Asn-Asn-Pro-Gln-Ile-Arg-CONH2
NH2-YWDANNPQIR-CONH2
化合物7(涉及SEQ ID NO:7):
NH2-Tyr-Trp-Asp-His-Ala-Asn-Pro-Gln-Ile-Arg-CONH2
NH2-YWDHANPQIR-CONH2
化合物8(涉及SEQ ID NO:8):
NH2-Tyr-Trp-Asp-His-Asn-Ala-Pro-Gln-Ile-Arg-CONH2
NH2-YWDHNAPQIR-CONH2
化合物9(涉及SEQ ID NO:9):
NH2-Tyr-Trp-Asp-His-Asn-Asn-Ala-Gln-Ile-Arg-CONH2
NH2-YWDHNNAQIR-CONH2
化合物10(涉及SEQ ID NO:10):
NH2-Tyr-Trp-Asp-His-Asn-Asn-Pro-Ala-Ile-Arg-CONH2
NH2-YWDHNNPAIR-CONH2
化合物11(涉及SEQ ID NO:11):
NH2-Tyr-Trp-Asp-His-Asn-Asn-Pro-Gln-Ala-Arg-CONH2
NH2-YWDHNNPQAR-CONH2
化合物12(涉及SEQ ID NO:12):
NH2-Tyr-Trp-Asp-His-Asn-Asn-Pro-Gln-Ile-Ala-CONH2
NH2-YWDHNNPQIA-CONH2
化合物13(涉及SEQ ID NO:13):
Ac-(D-Tyr)-Trp-Asp-His-Asn-Asn-Pro-Gln-Ile-Arg-CONH2
Ac-(D-Y)-WDHNNPQIR-CONH2
化合物14(涉及SEQ1 ID NO:14):
Ac-Tyr-Trp-Asp-His-Asn-Arg-Pro-Gln-Ile-Arg-CONH2
Ac-YWDHNRPQIR-CONH2
化合物15(涉及SEQ ID NO:15):
Ac-Tyr-Trp-Asp-His-Asn-Gln-Pro-Gln-Ile-Arg-CONH2
Ac-YWDHNQPQIR-CONH2
化合物16(涉及SEQ ID NO:16):
Ac-Tyr-Trp-Asp-His-Asn-Asp-Pro-Gln-Ile-Arg-CONH2
Ac-YWDHNDPQIR-CONH2
化合物17(涉及SEQ ID NO:17):
Ac-Tyr-Trp-Glu-His-Asn-Asn-Pro-Gln-Ile-Arg-CONH2
Ac-YWEHNNPQIR-CONH2
化合物18(涉及SEQ ID NO:18):
Ac-Tyr-Trp-Asp-His-Gln-Asn-Pro-Gln-Ile-Arg-CONH2
Ac-YWDHQNPQIR-CONH2
化合物19(涉及SEQ ID NO:19):
Ac-Tyr-Trp-Asp-His-Asn-Asn-Pro-Asn-Ile-Arg-CONH2
Ac-YWDHNNPNIR-CONH2
化合物20(涉及SEQ ID NO:20):
Ac-Tyr-Trp-Asp-His-Asn-Asn-Pro-Gln-Leu-Arg-CONH2
Ac-YWDHNNPQLR-CONH2
化合物21(涉及SEQ ID NO:21):
Ac-Tyr-Trp-Asp-His-Asn-Gln-Pro-Asn-Ile-Arg-CONH2
Ac-YWDHNQPNIR-CONH2
化合物22(涉及SEQ ID NO:22):
Ac-Tyr-Trp-Glu-His-Asn-Gln-Pro-Gln-Leu-Arg-CONH2
Ac-YWEHNQPQLR-CONH2。
附图说明
图1为使用本发明的多肽化合物1-12作用于LX-2细胞后,LX-2细胞中α-SMA的表达情况;其中以GAPDH作为内参。
图2为使用本发明的多肽化合物13-22作用于LX-2细胞后,LX-2细胞中α-SMA的表达情况;其中以GAPDH作为内参。
图3为在HFD诱导的NASH模型中,OGTT和ITT测试结果。
图4为在HFD诱导的NASH模型中,小鼠肝脏苏木精-伊红(H&E)染色病理切片图以及油红O染色病理切片图。
图5为在HFD诱导的NASH模型中,小鼠血清中甘油三酯、胆固醇、高密度脂蛋白、低密度脂蛋白、ALT和AST的变化情况。
图6为在CCl4诱导的肝脏纤维化模型中,小鼠肝脏H&E染色病理切片图;天狼星红染色病理切片图;以及α-SMA和Col-1α的免疫组化病理切片图。
图7为在CCl4诱导的肝脏纤维化模型中,小鼠血清中ALT和AST的变化情况。
具体实施方式
以下实施例是便于更好地理解本发明,但并不用于限定本发明。下述实施例中的实验方法,如无特殊说明,均为常规方法。
实验结果采用GraphPad Prism软件分析,数据以平均值±标准误差(Mean±SEM)表示,并通过t检验进行评价。模型组与对照组比较,以*P<0.05,**P<0.01,***P<0.001表示,给药组与模型组比较,以#P<0.05,##P<0.01,###P<0.001表示。Western blot结果采用Tanon图像分析软件进行分析。
下述实施例中所用的材料、试剂等,如无特殊说明,均可通过商业途径得到。
下述实施例中的小鼠均为健康雄性C57BL/6J小鼠,6-8周大,购自北京维通利华实验动物技术有限公司。
下述实施例中的多肽化合物1-22溶液均采用无菌的PBS作为溶剂配制。
实施例1、多肽化合物1-22的制备:
多肽化合物1-22采用Fmoc固相多肽合成法,由羧基端向氨基端方向合成,氨基酸连接顺序按照上述描述,依次连接。具体步骤如下:
①制备条件:采用MBHAR(Amide-MBHA-Resin酰胺保护的MBHA树脂)或者王树脂,以活化脂的方法,脱Fmoc保护基合成。
②茚检试剂:
试剂1:20g苯酚/5mL乙醇;
试剂2:0.05mL 0.001M KCN(水)/2.5mL吡啶;
试剂3:0.5g茚三酮/10mL乙醇;
茚检试剂的使用,试剂1:试剂2:试剂3=l:2:l(滴),沸水中加热3-10min。
③过程:
A.处理树脂:称取一定量MBHA树脂(1%交联度,200-400目,取代值0.5mmol/g)于合成仪中,加入DCM搅拌30min,抽干,再用DMF洗4次,每次2min,挑取少量树脂进行茚检,若树脂颜色无改变,表明正常,可使用。
B.脱Fmoc保护:20%哌啶(溶于DMF中)洗4次,每次2min,然后用DMF洗4次,每次2min,在第3次洗之后茚检,茚检显蓝紫色表明Fmoc已脱,可接下一个氨基酸。
C.缩合反应:将氨基酸、HOBT、HBTU依次加入烧杯中,加入DMF使其溶解,DMF体积尽量小,加入DIEA启动反应后,立即加入到合成仪中,之后用少量DMF冲洗烧杯和合成仪壁(投料量=树脂取代值(mmol/g)×m(树脂质量g)×M(肽的分子量g/mol)×几倍过量,氨基酸、HBTU、HOBT加入3倍过量,DIEA为液体碱,起到活化酯反应,加入6倍过量,保证每步接近99%成功)。整个反应体系用氩气保护。反应搅拌1h,抽干,用DMF洗3次,每次2min,茚检无改变,说明氨基酸已经接到树脂上。
D.每接一个氨基酸,都重复B、C,直至接肽完成为止。
E.多肽切割:
a.全部连接后,用20%哌啶脱F-moc保护,洗4次,每次2min,然后用DMF洗4次,每次2min,茚检显色后,用DCM洗2次,每次3min,甲醇洗1次,每次3min,再用DCM洗1次,每次3min,甲醇洗2次,每次3min。经过处理后,移开搅拌棒,用胶塞密封合成仪,再使用真空泵彻底抽干,至少2h以上,直至树脂完全干燥呈粉末状。
b.在抽干的合成仪中加入切割剂(TFA:Tis:水=95:2.5:2.5(V/V/V)),反应3h,每20min搅拌1min,用圆底烧瓶收集切割剂,并用TFA洗涤2次,每次5min(5mL/次),收集的TFA与之前收集的切割剂合并。
c.将所得的切割剂在旋转蒸发仪上减压抽干,把预先冷却的乙醚(50-80mL)加入到圆底烧瓶中,用力摇晃进行沉淀,静置。待白色固体沉淀到底部,使用滴管吸取上清液,加入分液漏斗中,补加等体积水,摇晃(中间放气几次),静置。这时,分液漏斗内部的液体会进行分层,把下层水相流入圆底烧瓶中,将沉淀进行溶解(如果不好溶解,可加醋酸调节溶解性)。
d.轻轻摇晃圆底烧瓶(防止乳化),使得溶解彻底,然后倒入分液漏斗,把水相流入烧杯收集,多洗几次,每次收集水相。
e.用保鲜膜密封烧杯,插孔、标记,-80℃过夜存放,然后真空冷冻干燥处理。
(2)粗肽脱盐
由于粗肽中存在反应过程中生成的副产物、盐和一些其他杂质,因此要对粗肽进行脱盐处理,初步除去杂质。首先称取一定量的sephadex G-25(约50-100目),加入5-10倍去离子水,加热溶胀3h,装柱。待自然沉降完全后,用10%醋酸平衡柱子2h左右,然后上样、10%醋酸进行洗脱,收集核酸蛋白紫外检测仪220nm处有吸收峰的溶液,同样用保鲜膜密封烧杯,插孔、标记,-80℃过夜存放,然后真空冷冻干燥处理。
(3)脱盐产物的纯化和纯度分析
用高效液相色谱进行纯化:制备柱为C18反相制备柱,规格为XBridgeTMBEH130Prep C18(10μm,19×250mm),洗脱体系为20%-80%乙腈/水/0.1%三氟乙酸,持续60min,流速:8mL/min,收集主峰。待真空冷冻干燥后,送质谱(ESI-MS)进行验证。
纯化产物的纯度分析:分析柱为C18反相制备柱,规格为XBridgeTMBEH130 PrepC18(10μm,4.6×250mm),洗脱体系为10%-90%乙腈/水/0.1%三氟乙酸,持续30min,流速:1mL/min,根据220nm色谱图积分,计算纯度。
合成的多肽采用高效液相色谱进行纯化(纯度>95%),并通过高分辨质谱进行表征。
基于以上合成步骤,合成本发明的如下表1所示的多肽化合物:
表1本发明的多肽化合物
实施例2、多肽化合物1-22改善和治疗非酒精性脂肪肝炎及肝纤维化的活性:
2.1多肽化合物1-22的体外细胞实验:
通过构建细胞模型—人肝星状细胞(LX-2)纤维化模型,对多肽化合物1-22活性成分进行初步测定。
LX-2细胞贴壁生长24h之后,实验组加入100μM多肽化合物1-22,对照组加入同等剂量的PBS,作用48h之后,提取细胞总蛋白,进行Western Blot实验检测α-平滑肌细胞(α-smooth muscle actin,α-SMA)蛋白的表达水平,结果如图1至图2所示。
图1的结果表明,对多肽化合物进行N端乙酰化修饰并不会影响多肽化合物的活性。丙氨酸扫描的结果表明多肽化合物的氨基酸序列很保守,仅替换第6位的氨基酸为Ala对多肽化合物的活性影响不大,可用于后续的活性优化。
图2的结果表明,在进一步的氨基酸替换实验结果中,仅将第6位氨基酸由Asn替换为Gln多肽化合物的活性增加,其他的氨基酸替换策略均会导致多肽化合物活性的降低。由此可见,在体外的细胞实验中,多肽化合物1-22均能体外抑制人肝星状细胞(LX-2)活化,其中化合物14-22的抑制效果更加明显。
2.2多肽化合物15的体内动物实验:
以多肽化合物15为例通过两种动物疾病模型(非酒精性脂肪性肝炎模型、肝脏纤维化模型)进行体内药效评估。
2.2.1多肽化合物15对高脂饲料(HFD)诱导的NASH模型的治疗作用:
(1)NASH模型的建立:
从中山大学实验动物中心购买雄性C57BL/6小鼠,体重20-22g。在温度25℃,12h/12h的明暗交替环境下饲养,适应环境1周后,开始喂养HFD饲料(D12492,Research Diets),对照组喂养正常的对照饲料(D12450B,Research Diets,RCD),喂养12周后,随机取出几只小鼠,取材,检测造模情况。确认造模成功之后,随机分成RCD组、HFD组、多肽化合物15组,按照500ug/kg腹腔注射药物多肽化合物15,RCD组、HFD组腹腔注射同等体积的PBS,每天给药一次,给药4周后取材。
(2)在HFD诱导的NASH模型中,腹腔注射多肽化合物15对小鼠非酒精性脂肪性肝炎(NASH)的作用:
HFD诱导的NASH模型,其表现特征为:肥胖,胰岛素抵抗,高糖血症,血脂异常和肝脏脂肪空泡样变性等。在多肽化合物15治疗HFD小鼠3周和4周的时候,分别进行口服葡萄糖耐受实验(OGTT)和胰岛素耐受测试(ITT),图3的实验结果显示,与HFD组相比,多肽化合物15组很好的改善了HFD小鼠口服葡萄糖耐受能力及胰岛素抵抗。图4的苏木精-伊红(H&E)染色结果显示,HFD组存在大片空泡区域,与之相比,多肽化合物15组空泡数目明显减少。在油红O染色中,能更直观显示出HFD组有大面积的脂滴存在于空泡内,药物处理之后,脂滴数目明显减少。血清学检测(图5)显示,对比HFD组,多肽化合物15处理之后,ALT和AST均下降,说明多肽化合物15能改善肝脏损伤。此外,多肽化合物15也可以明显降低HFD喂养小鼠血清中甘油三酯、胆固醇以及低密度脂蛋白的含量。
由以上结果可知:多肽化合物15可显著改善葡萄糖耐受和胰岛素抵抗,减少肝脏脂肪变性和非酒精性脂肪性肝炎。
其中,使用到的实验方法如下:
A.OGTT:
小鼠过夜禁食后,腹腔注射化合物1和5(500ug/kg)或者同等体积的PBS。4h之后,按照2g/kg灌胃葡萄糖(浓度为400mg/mL),在灌胃葡萄糖后,小鼠尾巴尖部取血,检测0、15、30、60和120min的血糖水平。
B.ITT:
OGTT测完一周之后,小鼠禁食5h,然后腹腔注射化合物1和5(500ug/kg)或者同等体积的PBS,并记录此时的血糖水平。1h后,按照0.25U/kg腹腔注射胰岛素,记录0、15、30、60和120min的血糖水平。
C.H&E染色:
1)烘片
取石蜡包埋的组织切片,60℃烘1h;
2)脱蜡、水化:
二甲苯20分钟→二甲苯20分钟→无水酒精15分钟→无水酒精15分钟→95%酒精10分钟→90%酒精5分钟→80%酒精5分钟;
3)染色:
苏木精7分钟→自来水冲洗干净→1%盐酸乙醇分化1s→自来水冲洗→伊红染色15s-20s→自来水冲洗;
4)脱水透明:
75%酒精1s→85%酒精1s→95%酒精1s→100%酒精1s→二甲苯→二甲苯;
5)封片:
晾干30min,树胶封片。
PAS染色:
流水冲3min→高碘酸氧化5min→双蒸水涮12下→Schiff试剂滴染15min(切片变成微粉红)→自来水冲洗5min→苏木素1min→水洗5min→脱水,干燥,封片。
2.2.2多肽化合物15对CCl4诱导的肝纤维化模型的治疗作用:
(1)肝纤维化模型的建立:
从中山大学实验动物中心购买雄性C57BL/6小鼠,体重20-22g。在温度25℃,12h/12h的明暗交替环境下饲养,适应环境1周后,开始注射CCl4(按照1:4溶解在玉米油中),对照组注射玉米油(Oil),腹腔注射3周后,随机取出几只小鼠,取材,观察造模情况。确认造模成功之后,随机分成Oil组、CCl4组、多肽化合物15组,按照500ug/kg腹腔注射药物化合物15,Oil组和CCl4组分别注射同等体积的PBS,每天给药一次,治疗3周后取材。
(2)在CCl4诱导的肝纤维化模型中,腹腔注射化合物15对治疗小鼠肝脏纤维化的作用:
CCl4诱导的肝纤维化模型,其表现特征为:肝脏组织出现局部的炎症及细胞坏死,以及大片纤维化区域等。在多肽化合物15治疗CCl4诱导的小鼠3周后,我们分别取了小鼠血液及肝脏用于血清学指标检测和病理学分析。由图6的H&E染色结果显示,相比Oil组,CCl4组出现明显的细胞坏死,及炎性细胞浸润,多肽化合物15组有明显改善。此外,还进行了天狼星红染色,用以标记纤维化区域,图6天狼星红的染色结果显示CCl4组有大片纤维化区域,多肽化合物15组有很好的改善纤维化的作用。图6中CD68的免疫组化染色结果也进一步确认了多肽化合物15具有良好的抗炎活性。图7的结果显示,与CCl4组相比,小鼠经多肽化合物15处理之后,血清中的ALT和AST均明显下降,说明本发明的多肽化合物能明显改善由CCl4引起的肝脏损伤。
综合上述结果,本发明的多肽化合物能显著改善和治疗肝纤维化以及肝脏损伤具有很好的肝保护作用,且改善和治疗效果显著。
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<110> 中山大学
<120> 多肽化合物在制备用于预防或治疗非酒精性脂肪肝病药物中的应用
<130> GD1899-21P125290
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Claims (8)
1.一类多肽化合物在制备用于预防或治疗非酒精性脂肪肝病药物中的应用,所述多肽化合物具有以下氨基酸序列表示的母体肽:
R1-Xaa1-Xaa2–Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-COR2
其中,R1=-NH2或-Ac;R2=-NH2或-OH;
Xaa1=Tyr,Ala或D-Tyr;
Xaa2=Trp或Ala;
Xaa3=Asp,Ala或Glu;
Xaa4=His或Ala;
Xaa5=Asn,Ala或Gln;
Xaa6=Asn,Ala,Arg,Gln或Asp;
Xaa7=Pro或Ala;
Xaa8=Gln,Ala或Asn;
Xaa9=Ile,Ala或Leu;
Xaa10=Arg或Ala;
当所述母体肽的氨基酸序列为R1-Tyr-Trp-Asp-His-Asn-Asn-Pro-Gln-Ile-Arg-COR2时,R1≠-NH2。
2.根据权利要求1所述的应用,其特征在于,所述多肽化合物具有与亲脂性的取代基、和/或蛋白、抗体相连的衍生物及结构。
3.根据权利要求1所述的应用,其特征在于,所述母体肽的氨基酸序列选自SEQ ID NO:2,SEQ ID NO:3,SEQ ID NO:4,SEQ ID NO:5,SEQ ID NO:6,SEQ ID NO:7,SEQ ID NO:8,SEQID NO:9,SEQ ID NO:10,SEQ ID NO:11,SEQ ID NO:12,SEQ ID NO:13,SEQ ID NO:14,SEQID NO:15,SEQ ID NO:16,SEQ ID NO:17,SEQ ID NO:18,SEQ ID NO:19,SEQ ID NO:20,SEQID NO:21和SEQ ID NO:22中的一种。
4.根据权利要求3所述的应用,其特征在于,所述母体肽的氨基酸序列选自SEQ ID NO:14,SEQ ID NO:15,SEQ ID NO:16,SEQ ID NO:17,SEQ ID NO:18,SEQ ID NO:19,SEQ IDNO:20,SEQ ID NO:21和SEQ ID NO:22中的一种。
5.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:非酒精性脂肪样变性、非酒精性脂肪肝炎、肝纤维化、胆汁淤积性肝硬化、肝纤维化合并的肝硬化。
6.一种组合物,其包含权利要求1至5任一项所述的多肽化合物。
7.根据权利要求6所述的组合物,其特征在于,所述组合物为药物组合物,还包含药学上可接受的载体或辅料。
8.根据权利要求7所述的组合物,其特征在于,所述组合物为片剂、胶囊、糖衣片剂、粒剂、口服溶液、糖浆、用于皮肤表面的油膏和药贴、气雾剂、鼻喷剂以及用于注射的无菌溶液中的至少一种剂型。
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