CN115317589A - 脯氨酰羟化酶抑制剂及其应用 - Google Patents
脯氨酰羟化酶抑制剂及其应用 Download PDFInfo
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- CN115317589A CN115317589A CN202211076173.2A CN202211076173A CN115317589A CN 115317589 A CN115317589 A CN 115317589A CN 202211076173 A CN202211076173 A CN 202211076173A CN 115317589 A CN115317589 A CN 115317589A
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- iron
- hydroxyproline
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- prolyl hydroxylase
- ferrous
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Abstract
本发明公开了一种脯氨酰羟化酶抑制剂以及其在铁缺乏及相关疾病预防和治疗中的应用。具体是将胶原蛋白消化降解产生的含羟脯氨酸二/三肽用作脯氨酰羟化酶抑制剂,通过激活低氧诱导因子2α信号通路,上调肠上皮细胞中二价金属离子载体1、十二指肠细胞色素b、膜铁转运蛋白和亚铁氧化酶等铁吸收转运蛋白的表达,进而刺激机体铁吸收,可应用在预防和治疗铁缺乏及相关疾病的食品、保健食品和药品中。本发明所采用的脯氨酰羟化酶抑制剂来源于食物蛋白质,故与常规人工合成药物相比,具有无毒副作用的优点。本发明所述应用可作为铁营养干预的补充或替代疗法,能够弥补铁营养干预在安全性和有效性方面的不足,对炎症或肾损伤引起的功能性铁缺乏尤为有效。
Description
技术领域
本发明涉及生物医药技术领域,具体涉及一种脯氨酰羟化酶抑制剂及应用。
背景技术
铁缺乏是营养不足最常见的一种类型,据全球疾病负担研究的调查显示,2017年铁缺乏影响了全球11亿人口(占营养不足人口总数61%),所致伤残调整寿命年(Disability-Adjusted Life-Years,DALYs)高达3000万人年,是造成5-14岁儿童DALYs的头号因素,是导致育龄妇女DALYs的第五大因素。
目前常见的口服铁剂和食物铁强化是应对铁缺乏的干预措施。但临床研究发现过量铁的摄入有着极大的健康风险,会造成肠道微生态功能紊乱,进而引起儿童腹泻和感染性疾病发生几率的上升。根据最新系统回顾和荟萃分析,口服铁剂对于大约30%孕期铁缺乏和20%学龄儿童铁缺乏没有预防效果。因此,有必要寻求一种有效和潜在安全的干预方式来替代或补充现有的干预措施。
机体铁稳态与各组织氧供应紧密相关,脯氨酰羟化酶(PHD)对HIFα亚基中脯氨酸残基的羟化作用是氧感应通路的核心部分。PHD抑制剂近年来已成为调控机体铁稳态的热点靶标,但天然来源PHD抑制剂尚未被充分地认识和利用。因此筛选天然来源的PHD抑制剂,通过调节铁稳态为铁缺乏的营养干预提供新思路。文献中未发现有关胶原蛋白肽抑制PHD的相关报道。
发明内容
本发明要解决的技术问题是提供一种天然脯氨酰羟化酶抑制剂,以及其在铁缺乏及相关疾病预防和治疗中的应用。
为解决上述技术问题,本发明采取的技术方案是:
本发明首次发现,羟脯氨酸寡肽,可以用作脯氨酰羟化酶抑制剂,尤其是羟脯氨酸二/三肽。
本发明提供的所述含羟脯氨酸二/三肽包括如下序列中的一种或多种:脯氨酰羟脯氨酸
(Pro-Hyp)、缬氨酰羟脯氨酸(Ala-Hyp)、谷氨酰羟脯氨酸(Glu-Hyp)、亮氨酰羟脯氨酸(Leu-Hyp)、丝氨酰羟脯氨酸(Ser-Hyp)、羟脯氨酰甘氨酸(Hyp-Gly)、缬氨酸-羟脯氨酸-甘氨酸(Ala-Hyp-Gly)、谷氨酸-羟脯氨酸-甘氨酸(Glu-Hyp-Gly)、脯氨酸-羟脯氨酸-甘氨酸(Pro-Hyp-Gly)、丝氨酸-羟脯氨酸-甘氨酸(Ser-Hyp-Gly)、甘氨酸-脯氨酸-羟脯氨酸(Gly-Pro-Hyp)和甘氨酸-羟脯氨酸-羟脯氨酸(Gly-Hyp-Hyp)。
本发明所述含羟脯氨酸二/三肽为胶原蛋白消化降解产生,胶原蛋白消化降解包括蛋白酶消化、酸水解或碱水解中的任一种或两种以上。
蛋白酶消化使用的蛋白酶包括胃蛋白酶、胰蛋白酶、碱性蛋白酶、木瓜蛋白酶、生姜蛋白酶、菠萝蛋白酶、无花果蛋白酶、中性蛋白酶中的任一种或两种以上。
本发明所述的脯氨酰羟化酶抑制剂可以用来制备预防和治疗铁缺乏及相关疾病的食品、保健食品和药品。
进一步的,本发明还提供一种预防和治疗铁缺乏及相关疾病的药物,以所述脯氨酰羟化酶抑制剂为活性成分,并与铁剂组合使用。
更进一步的,所述铁剂选自硫酸亚铁、乳酸亚铁、葡萄糖酸亚铁、富马酸亚铁、右旋糖酐铁、琥珀酸亚铁、多糖铁复合物、柠檬酸铁铵、柠檬酸铁、氯化高铁血红素、焦磷酸铁、铁卟啉、甘氨酸亚铁、还原铁、乙二胺四乙酸铁钠、羰基铁粉、碳酸亚铁、柠檬酸亚铁、延胡索酸亚铁、血红素铁、电解铁中的一种或两种以上。
本发明提供的药物可以预防和治疗的铁缺乏及相关疾病为铁缺乏症、缺铁性贫血、肾性贫血、运动性贫血、慢性病贫血、肿瘤相关性贫血和失血性贫血。
所述药物的给药途径为口服给药或胃肠外给药。所述胃肠外给药方式为静脉注射、皮下注射、肌肉注射、腹腔注射和局部注射中的一种。
本发明所述的脯氨酰羟化酶包括脯氨酰羟化酶结构域蛋白1(PHD1)、脯氨酰羟化酶结构域蛋白2(PHD2)和脯氨酰羟化酶结构域蛋白3(PHD3)中的任一种或两种以上。
本发明实验数据表明,胶原蛋白消化降解产生的含羟脯氨酸二/三肽可以通过螯合辅酶铁核和形成空间位阻两种方式,对脯氨酰羟化酶产生竞争性和非竞争性混合抑制作用,具有与化学合成PHD抑制剂相当的活性。其次,通过实验还进一步验证了胶原蛋白消化降解产生的含羟脯氨酸二/三肽在结肠癌细胞和大鼠十二指肠中激活了低氧诱导因子2α信号通路,通过上调二价金属离子载体1、十二指肠细胞色素b、膜铁转运蛋白和亚铁氧化酶等铁吸收转运蛋白的表达,提升了肠道细胞还原、吸收和转运铁的能力,同时能够缓解肾损伤造成的功能性铁缺乏,从而实现对铁缺乏及相关疾病的预防和治疗效果。
与现有技术相比,本发明的技术优势在于:
1)将胶原蛋白消化降解产生的含羟脯氨酸二/三肽用作脯氨酰羟化酶抑制剂是本发明首创,采用食物中天然存在的蛋白质——胶原蛋白获取了脯氨酰羟化酶抑制剂,因此,与常规人工合成脯氨酰羟化酶抑制剂相比,本发明所述脯氨酰羟化酶抑制剂预期不具备毒副作用;
2)实验数据表明,胶原蛋白消化降解产生的含羟脯氨酸二/三肽可以通过螯合辅酶铁核和形成空间位阻两种方式,对脯氨酰羟化酶产生竞争性和非竞争性混合抑制作用,具有与化学合成PHD抑制剂相当的活性,例如,Pro-Hyp抑制PHD3的EC50值和Ki值分别可达10.62μM和6.73μM。其次,通过实验还进一步验证了胶原蛋白消化降解产生的含羟脯氨酸二/三肽在结肠癌细胞和大鼠十二指肠中激活了低氧诱导因子2α信号通路,通过上调二价金属离子载体1、十二指肠细胞色素b、膜铁转运蛋白和亚铁氧化酶等铁吸收转运蛋白的表达,提升了肠道细胞还原、吸收和转运铁的能力,从而实现对铁缺乏及相关疾病的预防和治疗效果;因此,胶原蛋白消化降解产生的含羟脯氨酸二/三肽可以作为一种脯氨酰羟化酶抑制剂而刺激机体自身肠道铁吸收,相比于传统铁营养干预,在解决炎症或肾损伤引起的功能性铁缺乏方面具有显著的应用优势;
3)实验表明,胶原蛋白消化降解产生的含羟脯氨酸二/三肽相当耐受消化道和血液肽酶降解,故可以口服给药,考虑到蛋白质很少通过该途径给药的事实,这是本发明的一个显著优点。
附图说明
下面结合附图对本发明的具体实施方式和有益效果作进一步详细的说明。
图1是重组人PHD3酶促反应动力学:(a)底物浓度依赖性;(b)Pro-Hyp浓度的影响(底物浓度为Km);(c)不同Pro-Hyp浓度时的双倒数图。
图2是PHD3分别与(a)Pro-Hyp和(b)HIF19的优势结合构象,以及二者在(c)蛋白填充模型和(d)球棍模型中合并后的构象。
图3是胶原蛋白降解的含羟脯氨酸二/三肽(CH)和Pro-Hyp对于肠上皮细胞HIF-2α信号通路的影响:HIF-2α蛋白水平的Western blotting(a)图像分析;(b)密度分析;(c)双荧光素酶报告基因法测定低氧反应元件(HRE)活性;(d)HIF-2α转录水平。
图4是胶原蛋白降解的含羟脯氨酸二/三肽(CH)和Pro-Hyp对于肠道铁稳态相关蛋白的表达的影响:(a)二价金属离子转运蛋白(DMT1);(b)十二指肠细胞色素b(Dcytb);(c)铁转运蛋白(FPN);(d)亚铁氧化酶(HEPH)。
图5是胶原蛋白降解的含羟脯氨酸二/三肽(CH)和Pro-Hyp对于肠道铁吸收的促进作用:(a)Caco-2单层细胞铁吸收的动力学;(b)上清培养液中积累的二价铁浓度;(c)Transwell铁转运装置中经单层细胞转运到下室的铁浓度。
图6是口服胶原蛋白降解的含羟脯氨酸二/三肽(CH)或Pro-Hyp对大鼠十二指肠铁转运蛋白mRNA表达水平的影响:(a)二价金属离子载体1(DMT1)和十二指肠细胞色素b(Dcytb);(b)铁转运蛋白(FPN)和亚铁氧化酶(HEPH);(c)HIF-2α的Western blotting图像;(d)密度分析。
图7是胶原蛋白降解的含羟脯氨酸二/三肽(CH)对于肾性贫血的改善作用:(a)肾脏指数;(b)尿液中尿素氮含量;(c)血液中血红蛋白含量;(d)血液中红细胞数量。
具体实施方式
实施例1
胶原蛋白来源的含羟脯氨酸二/三肽的脯氨酰羟化酶抑制活性评估及机理分析,具体实验过程和实验结论如下:
图1是重组人PHD3酶促反应动力学:(a)底物浓度依赖性;(b)Pro-Hyp浓度的影响(底物浓度为Km);(c)不同Pro-Hyp浓度时的双倒数图。
图2是PHD3分别与(a)Pro-Hyp和(b)HIF19的优势结合构象,以及二者在(c)蛋白填充模型和(d)球棍模型中合并后的构象
利用体外重组的PHD3,测定脯氨酰羟脯氨酸(Pro-Hyp)对其的抑制活性。将PHD3基因插入到pET-32a载体的EcoRV和SalI位点之间,构建质粒pET-32a-PHD3。质粒的表达宿主为大肠杆菌,经热休克(42℃,30s)连接质粒转化,在含有氨苄青霉素的标准琼脂平板上选择pET-32a-PHD3单个菌落接入LB肉汤中,在37℃摇床培养过夜,在600nm处的光密度达到0.6-0.8之间。然后用200μL IPTG诱导,在25℃摇床中培养4h,使PHD3在大肠杆菌中过表达。采用镍(Ni)亲和色谱纯化,大肠杆菌11000×g离心9min,重悬于8mL预冷的结合缓冲液(pH7.9的20mM Tris-HCl缓冲液,含0.5M NaCl和5mM咪唑)中,在冰上超声处理后于14000×g离心20min。离心后的上清液以20mL/h的流速,上样到2.5mL的Ni-NTA琼脂糖柱上。吸附重组PHD3后,用10倍柱体积的结合缓冲液洗涤柱。然后用6倍柱体积的洗脱缓冲液(pH 7.9的20mM Tris-HCl缓冲液,含0.5M NaCl和1M咪唑)洗脱重组的PHD3。得到的PHD3在含有2MNaCl和5mM DTT的磷酸钠缓冲液(20mM,pH 7.0)中透析两次,以除去咪唑。PHD属于2-酮戊二酸(2-OG)依赖性加氧酶超家族,Fe2+与酶结合形成Fe2+-酶复合物,依次结合2-OG和HIFα亚基,因此,可以根据2-OG的消耗情况判断酶反应的进程。整个反应体系,底物为HIF 19肽(序列为DLDLEMLAPYIPMDDDFQL)。进行抑制实验时,向整个体系中加入不同浓度的Pro-Hyp,通过向反应混合物中加入4μL 2-OG(160mM)引发反应。通过在Synergy H4荧光酶标仪(Bio-Tek)(340nm激发;420nm发射)检测2-OG的衍生化产物的荧光强度来定量催化反应之后剩余的2-OG的浓度。
图1a可以看出,合成的重组人PHD3蛋白米氏常数为2.15±0.22μM,与文献报道的2.9±0.4μM接近;Pro-Hyp能够抑制重组人PHD3蛋白的活性,其半效浓度EC50为10.62μM(图1b),抑制常数Ki为6.73μM(图1c),与文献中化学合成PHD抑制剂的活性相当。因此Pro-Hyp能够有效的抑制PHD的活性,进而稳定HIF的水平。
进一步利用分子模拟技术研究了胶原来源含羟脯氨酸二/三肽抑制PHD的可能机制。以PHD3(EGLN1,protein Data Bank ID:2G19)的晶体结构为模板,去除游离水和非极性氢,加入Gasteiger电荷和部分原子电荷,并使用MGLTools 1.5.6软件(The ScrippsResearch Institute,CA,USA)中的AutoDockTools分配脱溶参数,通过同源建模模拟PHD3(EGLN3)的三维蛋白结构。用ChemDraw19.0构建了胶原蛋白来源的典型二/三肽和Roxadustat的三维结构,并用Chem3D19.0(PerkinElmer,MA,USA)进行了能量最小化。用Autodock 4.2.6软件将胶原蛋白源的二/三肽和Roxadustat与PHD3进行分子对接,采用半柔性对接方法,使配体可以灵活,受体骨架原子在计算中保持刚性。以PHD3活性位点上的铁(II)原子为中心,构建了网格点间距为 的高分辨率矩形网格框从100种拉马克遗传算法对接解的最低结合能簇中选择最优对接构型。配体的初始位置为随机设置,其余对接参数为默认设置。对接结果使用PyMOL程序(NY,USA)进行可视化。Pro-Hyp在PHD3活性位点上的疏水口袋拟合度良好,其中的脯氨酸的亚氨基N原子和酰胺O原子与PHD3活性口袋内的铁核形成配位键(图2a),其羟脯氨酸残基与酶的Y125、R205和T209残基之间的氢键(图2b),Pro-Hyp中疏水基团与PHD3活性口袋内Y132、V198,W211,M121等疏水性氨基酸残基之间形成较好的疏水性接触(图2c)。显然,Pro-Hyp可以在一定程度上作为铁螯合剂阻断PHD3的活性位点,这解释了其非竞争性抑制作用(图2c)。HIF19肽中P9的酰胺O原子与PHD3活性口袋内的R144形成氢键,引导P9靠近铁离子发生羟化反应。将PHD3分别与Pro-Hyp和HIF19的对接模型在蛋白填充模型(图2c)和球棍模型(图2d)中进行合并后发现,Pro-Hyp会对HIF19肽中P9靠近铁离子造成空间位阻,表明Pro-Hyp与HIF19肽之间存在竞争性拮抗关系,Pro-Hyp可以竞争性地和立体地阻碍底物对酶活性中心的结合,这解释了其竞争性抑制作用(图2c)。
如表1所示,据预测,Pro-Hyp的结合能量值与口服的PHD抑制剂Roxadustat的结合能量值接近(表1),表明Pro-Hyp作为膳食来源的PHD抑制剂具有很大的潜力。同时,计算了脯氨酰羟脯氨酸(Pro-Hyp)、缬氨酰羟脯氨酸(Ala-Hyp)、谷氨酰羟脯氨酸(Glu-Hyp)、亮氨酰羟脯氨酸(Leu-Hyp)、丝氨酰羟脯氨酸(Ser-Hyp)、羟脯氨酰甘氨酸(Hyp-Gly)、缬氨酸-羟脯氨酸-甘氨酸(Ala-Hyp-Gly)、谷氨酸-羟脯氨酸-甘氨酸(Glu-Hyp-Gly)、脯氨酸-羟脯氨酸-甘氨酸(Pro-Hyp-Gly)、丝氨酸-羟脯氨酸-甘氨酸(Ser-Hyp-Gly)、甘氨酸-脯氨酸-羟脯氨酸(Gly-Pro-Hyp)和甘氨酸-羟脯氨酸-羟脯氨酸(Gly-Hyp-Hyp)与PHD的结合能量值,发现羟脯氨酸残基的存在可以大幅提升二/三肽与PHD3的结合。以上体外实验表明,因此,胶原蛋白消化降解产生的含羟脯氨酸二/三肽可以通过螯合辅酶铁核和形成空间位阻两种方式,对脯氨酰羟化酶产生竞争性和非竞争性混合抑制作用,具有与化学合成PHD抑制剂相当的活性。
表1.通过分子对接计算胶原蛋白来源的含羟脯氨酸二/三肽和Roxadustat对人PHD3蛋白的预测结合能量值
配体 | 结合能量值(kcal/mol) |
Ala-Hyp | -7.48 |
Glu-Hyp | -7.05 |
Leu-Hyp | -7.69 |
Ser-Hyp | -7.39 |
Pro-Hyp | -7.91 |
Hyp-Gly | -7.06 |
Gly-Pro-Hyp | -8.33 |
Ala-Hyp-Gly | -8.80 |
Glu-Hyp-Gly | -9.00 |
Pro-Hyp-Gly | -9.42 |
Ser-Hyp-Gly | -7.85 |
Gly-Hyp-Hyp | -8.58 |
Roxadustat | -8.11 |
实施例2
胶原蛋白降解的含羟脯氨酸二/三肽(CH)及Pro-Hyp在体外Caco-2肠上皮细胞中对PHD的抑制作用及对铁吸收转运的调控作用,具体实验过程和实验结论如下:
图3胶原蛋白降解的含羟脯氨酸二/三肽(CH)和Pro-Hyp对于肠上皮细胞HIF-2α信号通路的影响:HIF-2α蛋白水平的Western blotting(a)图像分析;(b)密度分析;(c)双荧光素酶报告基因法测定低氧反应元件(HRE)活性;(d)HIF-2α转录水平。
图4胶原蛋白降解的含羟脯氨酸二/三肽(CH)和Pro-Hyp对于肠道铁稳态相关蛋白的表达的影响:(a)二价金属离子转运蛋白(DMT1);(b)十二指肠细胞色素b(Dcytb);(c)铁转运蛋白(FPN);(d)亚铁氧化酶(HEPH)。
图5胶原蛋白降解的含羟脯氨酸二/三肽(CH)和Pro-Hyp对于肠道铁吸收的促进作用:(a)Caco-2单层细胞铁吸收的动力学;(b)上清培养液中积累的二价铁浓度;(c)Transwell铁转运装置中经单层细胞转运到下室的铁浓度。
本实验选用结构功能类似于小肠上皮细胞的结肠癌细胞Caco-2,实验所用细胞株购自中国科学院典型培养物保藏委员会细胞库。细胞使用含10%胎牛血清、1%青霉素-链霉素-两性霉素B混合溶液的DMEM高糖培养基于25cm2细胞培养瓶中培养,培养瓶置于37℃、5%CO2恒定湿度的细胞培养箱中,每隔2d更换培养基,当细胞的汇合度达到80%时,根据实验需要按一定比例进行铺板。将Caco-2细胞用高糖DMEM完全培养基稀释至一定浓度,以每孔2×105个细胞的密度将Caco-2悬液接种于6孔细胞培养板,每隔2d更换培养基,待细胞完全汇合后(约3-4d),每隔1d更换培养基,约10d左右细胞将完全分化成为单层,即得体外肠道细胞模型,随后加入各浓度下的胶原蛋白降解的含羟脯氨酸二/三肽(CH)、Pro-Hyp及三种胶原肽中特征性氨基酸孵育,并设置不加药物的培养基作为对照组,细胞在培养箱中培养24h后,吸去培养基后继续加入各浓度上述溶液孵育,总共作用于细胞的时间为48h,利用Western blotting研究不同组分对细胞HIF-2α表达及肠道铁稳态的影响。如图3a,3b的Western blotting结果所示,孵育48h后,CH及Pro-Hyp能够显著提高肠细胞HIF-2α的表达水平,其主要机制是抑制了PHD活性,而并非提高HIF-2α的转录水平(图3c)。之后,将含HIF-2α受体反应元件(HRE)片段的质粒通过Lipofectamine 3000导入Caco-2细胞中,利用双荧光素酶报告基因法测定了HIF-2α下游基因的转录活性,发现下游基因的转录活性随着Pro-Hyp浓度的增加呈现出剂量依赖性增加(图3d)。进一步检测了HIF-2α下游肠道铁稳态相关蛋白的表达,图4中可以看出,CH和Pro-Hyp对肠腔侧转运铁的二价金属离子载体1(DMT-1)和负责铁还原的十二指肠细胞色素b(Dcytb)的表达及肠道基底侧铁转运蛋白(FPN)和亚铁氧化酶(HEPH)的表达起到了显著的促进作用(P<0.05)。
为了进一步验证其对铁吸收的促进作用,利用钙黄绿素荧光法测定了细胞铁吸收动力学变化。利用Caco-2细胞模型模拟小肠近端pH值5.5的条件,以5×104个/孔的密度接种于胶原覆盖的24孔板,待细胞分化完全后,更换无血清MEM培养基进行饥饿处理24h,之后加入钙黄绿素乙酰氧基甲酯(Calcein-AM)在37℃孵育30分钟后,加入970μL含有MES(调节pH值5.5)的HBSS溶液,及包含1mM氨三乙酸-Fe3+的样品溶液,在Synergy H4荧光酶标仪(Bio-Tek)中37℃孵育30分钟,每3分钟记录一次钙黄绿素荧光(485nm激发,530nm发射)。Calcein-AM是一种可透过膜的非荧光分子,在细胞内被胞质酯酶裂解后发出荧光,可标记二价金属离子在细胞弱结合铁池中的含量。在Calcein荧光铁吸收实验中(图5a),可以看到经过CH孵育后的细胞铁吸收能力明显增强(P<0.01),经Pro-Hyp孵育后铁吸收能力也产生了一定的提高(P<0.05),为了研究细胞对于二价铁的还原能力,又使用了红菲绕啉二磺酸钠(BPDS),其可以快速结合Fe2+形成一种粉色络合物,可以通过的量判断细胞对Fe3+的还原能力。从图5b可以看出,CH和Pro-Hyp孵育过的细胞形成的络合物含量明显增加(P<0.05)。随后进一步验证从细胞基底侧转运出去的铁含量,构建了Transwell铁转运模型,通过测量下室的铁含量判断细胞铁吸收转运能力。图5c展示了Transwell铁转运实验的结果,可以看到经单层细胞转运到下室的铁含量结果与钙黄绿素铁吸收实验、亚铁积累实验结果相一致,经过CH与Pro-Hyp孵育后的细胞转运铁的能力明显增强(P<0.05)。因此表明胶原蛋白降解的含羟脯氨酸二/三肽能够通过抑制PHD活性,稳定HIF-2α,从而提高肠道铁稳态相关蛋白表达,促进肠道铁的转运与吸收。
实施例3
胶原蛋白降解的含羟脯氨酸二/三肽(CH)及Pro-Hyp对大鼠十二指肠PHD的抑制作用及铁吸收转运的调控作用,具体实验过程和实验结论如下:
图6口服胶原蛋白降解的含羟脯氨酸二/三肽(CH)或Pro-Hyp对大鼠十二指肠铁转运蛋白mRNA表达水平的影响:(a)二价金属转运蛋白1(DMT1)和十二指肠细胞色素b(Dcytb);(b)铁转运蛋白(FPN)和亚铁氧化酶(HEPH);HIF-2α的Western blotting(c)图像分析和(d)密度分析。
为了验证胶原蛋白降解的含羟脯氨酸二/三肽是否能在体内刺激肠道非血红素铁的吸收,选用21±3天周龄的雌性Sprague-Dawley大鼠,动物房温度24±2℃,湿度55±15%,早上6点开灯,12/12h明暗交替,在整个研究期间动物可自由摄食和饮用超纯水。适应1周后,随机分为5组,先将老鼠禁食12h(超纯水正常供应),以排除饮食干扰,然后按照高剂量1.2g/kg和低剂量0.6g/kg灌胃CH以及按照高剂量0.2g/kg和低剂量0.1g/kg灌胃Pro-Hyp,灌胃后2h,进行麻醉,取十二指肠、空肠,进行mRNA和蛋白的提取。测定十二指肠基因Dcytb、DMT1、FPN和HEPH的转录水平,如图6a和6b所示,CH和Pro-Hyp诱导的大鼠十二指肠Dcytb、DMT1、FPN和HEPH的基因转录水平呈剂量依赖性增加,证实了CH和Pro-Hyp在体内对肠道非血红素铁吸收的直接刺激作用。根据Western blotting结果(图6c和6d),CH和Pro-Hyp剂量依赖性地增加了大鼠十二指肠HIF-2α的蛋白水平,验证了CH在体内对HIF-2α信号通路的激活。
实施例4
胶原蛋白降解的含羟脯氨酸二/三肽对于肾损伤造成的功能性铁缺乏的改善作用评估,具体实验过程和实验结论如下:
图7胶原蛋白降解的含羟脯氨酸二/三肽(CH)对于肾损伤造成的功能性铁缺乏的改善作用:(a)肾脏指数;(b)尿液中尿素氮含量;(c)血液中血红蛋白含量;(d)血液中红细胞数量
缩写:胶原蛋白降解的含羟脯氨酸二/三肽高剂量组(CH-H),胶原蛋白降解的含羟脯氨酸二/三肽低剂量组(CH-L)。
6周龄C57BL/6小鼠,每组8只,动物房温度24±2℃,湿度55±15%,12/12h光照周期(早上8点至晚上8点照明),在整个研究期间动物可自由摄食和饮用超纯水。获得中国海洋大学动物实验伦理委员会批准(批准号:SPXY20220702)。
小鼠适应1周后,各组每日灌胃100μL 50mg/kg体重的腺嘌呤(0.5%羧甲基纤维素钠溶液中),进行造模,同时按照分组,每日灌胃剂量100μL胶原肽(0.5%羧甲基纤维素钠溶液中),高剂量为600mg/kg;低剂量为150mg/kg,Roxadustat组为100μL25 mg/kg(0.5%羧甲基纤维素钠溶液中),对照组为100μLPBS(0.5%羧甲基纤维素钠溶液中)造模加治疗共计四周。
如图7a所示,灌胃PBS组经过长时间的腺嘌呤造模损伤,肾脏明显肿大,肾脏指数较高,而CH后能够显著缓解肾脏的肿大(P<0.05)。腺嘌呤的存在引发导致肾功能损伤,大量的尿素氮无法从尿液中排出,因此在灌胃PBS组的尿液中的尿素氮显著降低(P<0.05)(图7b),而CH缓解了这一趋势。同时,CH能够提高血液中血红蛋白及红细胞数量(图7c,7d),改善了肾损伤所造成铁缺乏的问题,与阳性药物Roxadustat效果一致。因此,CH能够有效缓解肾功能损伤,对改善肾损伤引起的功能性铁缺乏尤为有效。
以上实施例显示和描述了本发明的基本原理和主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,而不是以任何方式限制本发明的范围,在不脱离本发明范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的范围内。
Claims (8)
1.胶原蛋白消化降解寡肽在脯氨酰羟化酶抑制剂上的应用。
2.根据权利要求1所述的胶原蛋白消化降解寡肽,其特征在于,为胶原蛋白消化降解产生的含羟脯氨酸二/三肽。
3.一种脯氨酰羟化酶抑制剂,其特征在于,所述抑制剂为胶原蛋白消化降解产生的含羟脯氨酸二/三肽。
4.根据权利要求3所述的脯氨酰羟化酶抑制剂,其特征在于,含羟脯氨酸二/三肽包括如下序列中的一种或多种:脯氨酰羟脯氨酸(Pro-Hyp)、缬氨酰羟脯氨酸(Ala-Hyp)、谷氨酰羟脯氨酸(Glu-Hyp)、亮氨酰羟脯氨酸(Leu-Hyp)、丝氨酰羟脯氨酸(Ser-Hyp)、羟脯氨酰甘氨酸(Hyp-Gly)、缬氨酸-羟脯氨酸-甘氨酸(Ala-Hyp-Gly)、谷氨酸-羟脯氨酸-甘氨酸(Glu-Hyp-Gly)、脯氨酸-羟脯氨酸-甘氨酸(Pro-Hyp-Gly)、丝氨酸-羟脯氨酸-甘氨酸(Ser-Hyp-Gly)、甘氨酸-脯氨酸-羟脯氨酸(Gly-Pro-Hyp)和甘氨酸-羟脯氨酸-羟脯氨酸(Gly-Hyp-Hyp)。
5.权利要求4所述的脯氨酰羟化酶抑制剂在制备预防和治疗铁缺乏及相关疾病的食品、保健食品和药品中的应用。
6.预防和治疗铁缺乏及相关疾病的药物,其特征在于,以权利要求4所述脯氨酰羟化酶抑制剂为活性成分,并与铁剂组合使用。
7.根据权利要求6所述的药物,其特征在于,所述铁剂选自硫酸亚铁、乳酸亚铁、葡萄糖酸亚铁、富马酸亚铁、右旋糖酐铁、琥珀酸亚铁、多糖铁复合物、柠檬酸铁铵、柠檬酸铁、氯化高铁血红素、焦磷酸铁、铁卟啉、甘氨酸亚铁、还原铁、乙二胺四乙酸铁钠、羰基铁粉、碳酸亚铁、柠檬酸亚铁、延胡索酸亚铁、血红素铁、电解铁中的一种或两种以上。
8.根据权利要求6所述的药物,其特征在于,所述可以预防和治疗的铁缺乏及相关疾病为铁缺乏症、缺铁性贫血、肾性贫血、运动性贫血、慢性病贫血、肿瘤相关性贫血和失血性贫血。
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