CN112759656A - 一种用于解除脑内神经元细胞铁胁迫的多肽 - Google Patents
一种用于解除脑内神经元细胞铁胁迫的多肽 Download PDFInfo
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Abstract
本发明公开了一种用于解除脑内神经元细胞铁胁迫的多肽，包括螯合铁序列和导肽序列，所述螯合铁序列、导肽序列均由氨酸残基组成，所述导肽序列为QSDIVAHAHLL，所述Q为谷氨酰胺残基简称、S为丝氨酸残基简称、I为异亮氨酸残基简称、V为缬氨酸残基简称、L为亮氨酸残基简称。本发明多肽可有效去除过量脑内冗余的铁，减少大脑中的自由基对脑组织的损伤，从而保护了大脑并减轻了认知能力下降。
Description
技术领域
本发明属于生物技术领域，具体涉及一种用于解除脑内神经元细胞铁胁迫的多肽，该多肽用于减少脑内自由基以保护神经元细胞。
背景技术
衰老引起脑内铁升高，导致神经细胞损伤，随疾病进展可表现为老年痴呆。2010年全球有3500万的AD患者，预计到2050将有1.13亿患者。目前全球每年的AD医疗费用估计有3150亿美元。目前，我国的AD患者总数约有900万。我国60岁以上老年人口现已超过2亿，预计2025年将为3.1亿。而超过60岁人口中的AD患病率估计在3％左右，85岁以上人口中的患病率估计在40％左右。
老年痴呆症(Alzheimer’s Disease，老年痴呆症)是一种神经退行性疾病，因神经元大量坏死，导致脑萎缩，临床表现以不同程度的智力障碍为主。伴有性格障碍，情感障碍，记忆障碍，行为障碍以及烦躁性神经系统症状和体征，临床有两个特征：一是为人性格改变，病人主观任性，自私狭隘，不喜欢与人交往，对家人缺乏感情及道德感多疑，严重时不能自理当众裸体甚至发生违法行为；二是记忆力障碍，以记忆力减退尤为显著，刚刚做完的事就会遗忘，出门时不能记住回家的路，认不出几日前见过的人。老年痴呆患者给家庭带来极大烦恼和负担，目前，治疗老年痴呆症的药有很多，这些药物大多只能暂时缓解患者认识功能减退，但不能延缓疾病发展，而且具有严重的肝肾毒性等不良反应，且价格昂贵。
血脑屏障(BBB,一种由内皮细胞紧密连接，可将脑组织与血液隔开的一种结构)会阻止分子量大于500Da的分子进入大脑，严重制约了大分子药物疗效，研究表明，生物体在新陈代谢生命活动过程中，会不断生成一些氧化活性物质，如H2O2等，可与Fe2+离子发生反应，催生自由基，生成的自由基可进一步与体内的生物大分子作用，破坏组织和细胞内的脂质、蛋白质等，导致细胞损伤，组织坏死；老年痴呆症患者的脑内铁含量比正常脑高出约35％，因此，除去脑内冗余的铁，是治疗老年痴呆症的一个方向，曾有人用去铁敏、地拉罗斯等口服药用于去除血液和脑内过高的铁以缓解病症，但久服这些药物易引起肾衰和血液病，存在严重的副作用。
发明内容
针对现有技术中存在的问题，本发明设计的目的在于提供一种用于解除脑内神经细胞铁胁迫的多肽。
本分明通过以下技术方案加以实现：
所述的一种用于解除脑内神经元细胞铁胁迫的多肽，其特征在于包括螯合铁序列和导肽序列，所述螯合铁序列、导肽序列均由氨酸残基组成，所述导肽序列为QSDIVAHAHLL，所述Q为谷氨酰胺残基简称、S为丝氨酸残基简称、I为异亮氨酸残基简称、V为缬氨酸残基简称、L为亮氨酸残基简称。
所述的一种用于解除脑内神经元细胞铁胁迫的多肽，其特征在于所述螯合铁序列包括若干个X序列，所述X序列由H、A、Y、E、D组成，所述H、A、Y、E、D任意排列，所述H为组氨酸残基，所述A为丙氨酸残基，所述Y为酪氨酸残基，所述E为谷氨酸残基，所述D为天冬氨酸残基。
所述的一种用于解除脑内神经元细胞铁胁迫的多肽，其特征在于所述导肽序列引螯合铁序列穿越血脑屏障解除神经细胞铁胁迫。
本发明多肽可有效去除过量脑内冗余的铁，减少大脑中的自由基对脑组织的损伤，从而保护了大脑并减轻了认知能力下降。
附图说明
图1为老化小鼠脑内富含铁和自由基，坏死神经元广分布图；
A)老化鼠的大脑比正常鼠的脑含铁更多。老化脑中的铁平均为0.72μg/g湿重，集中在AD脑的胼胝体、皮层、海马、视前外侧区、扣带回皮层和杏仁核，而在脑室、前庭区、内嗅皮层和侧脑区较少。正常脑只有0.43μg/g湿重。a)IG-RSGb；b)D3V；c)PtA；d)S1BF；e)LPMR；f)CA2-CA3；g)颞皮质；h)PRh-Lent；i)杏仁核；j)丘脑；k)下丘脑。B)衰老小鼠的脑中含大量·OH自由基，比正常脑约高140％。C)老化小鼠脑切片TUNEL-DAB染色表明，老化脑中广泛分布坏死的神经元(棕色斑点为坏死神经元)。*：p＜0.05；
图2为5-YHEDA亲铁性分析图；
A)合成的5-YHEDA质谱。m/z＝715.85峰是含5个H+的片段，为一重“YHEDA”。峰630.11是“YHED”寡肽，是“YHEDA”失去了丙氨酸和几个氢原子的碎片。峰894.81和1192.40分别是含4H+离子和3H+碎片，分别是“YHEDA-Y”和“YHEDA-YHE”片段。综合以上分析，整个肽的分子量为3574.5。B)与或未与FeCl3孵育的5-YHEDA肽的TEM图像。FeCl3孵育导致了5-YHEDA肽的团聚。X射线能谱显示，铁沿肽纤维共定位。C)等温滴定释热分析表明，Fe：5-YHEDA反应生成的复合物损失了91255kCal/mol的焓和8973kCal/mol的Gibbs，表明5-YHEDA与铁发生了结合。D)FeCl3孵育前后的5-YHEDA的红外光谱。720cm-1处的信号是Tyr残基中的苯-OH基；在2925cm-1处和
处的峰，可能是Glu或Asp残基中的-COOH。His残基中的C-N(2375cm-1)和C＝N
均被削弱，表明发生了铁结合。此外，代表主链丙烯酰胺NH伸展
C＝O键位移
NH弯曲
和CN伸展(1420)的信号
)发生红移，表明铁孵育后肽链骨架中发生了扭折。*：p＜0.05；
图3为5-YHEDA和bs-5YHEDA通过还原铁和羟基自由基保护细胞的体外实验图；
A)肽的YHEDA重复序列越多，则对铁和·OH自由基还原效果越明显。B)随着5-YHEDA浓度的增加，铁减少。尽管结合段“QSDIVAHLL”稀释了亲铁残基比份，可能会削弱其除自由基的能力，但实验结果显示5-YHEDA连接了LDLR结合段后，仍能有效地还原富铁培养基中的自由基。C)SH-sy5y细胞在富铁培养基中萎缩，树突棘(△)减少、轴突运输(↖)消失；相反，在高铁培养基中补充5-YHEDA或bs-5-YHEDA后，细胞存活增多，轴突和树突伸展、树突棘增加，并且细胞质转运活跃，沿树突可观察到运输囊泡。D、E)检测表明5-YHEDA和bs-5-YHEDA肽均可降低培养基中的·OH自由基和铁的含量，提高细胞存活率。*：p＜0.05；
图4为LDLR-5-YHEDA穿血脑屏障进入脑免疫共沉淀、免疫组化、免疫胶体金颗粒图；
A)在100Kd处，泳道3的LDLR上方有一个粗黑带，约为bs-5-YHEDA和LDLR分子量的总和，为bs-5-YHEDA：LDLR结合的复合物所在位置。而在泳道5中，未连接LDLR结合段的5-YHEDA和LDLR的混合物电泳，跑出两个独立的条带，一个与LDLR的水平位置一致，另一个跑到底部。而且在此泳道未发现大于100Kd的复合物条带，表明未连接LDLR结合段的5-YHEDA几乎不与LDLR相互作用。B)免疫组化显示，将LDLR结合片段连接到5-YHEDA后，该肽能够穿过BBB并进入大脑。a、b)对照和经5-YHEDA心注的脑切片均无染色。c)但是在注射了连有LDLR结合段的bs-5-YHEDA后的小鼠大脑中，有分散5-YHEDA免疫组化染色斑点，可能是bs-5-YHEDA越过BBB进入大脑后到达的位置；C)3H标bs-5-HAYED放射自显影显示，经心注200μl20μΜ 3H-bs-5-HAYED 4天后，大脑中残余的3H-bs-5-HAYED浓度约为0.25μM，仍保持活性。D)注射bs-5-YHEDA的老化小鼠脑血管周围检测到5-YHEDA免疫胶体金颗粒。铁簇聚与bs-5-YHEDA肽共定位，而无胶体金颗粒处的铁浓度较低。与此形成对比，在用无LDLR连接段的5-YHEDA处理的脑中，未观察到金颗粒，且铁背景更密；
图5为ubs-5-YHEDA可清除衰老小鼠大脑中的铁和自由基，防止神经元坏死和认知能力的下降示意图
为了评估5-YHEDA在保护大脑中的作用，阻止老化鼠认知恶化，我们将连有LDLR结合片段的5-YHEDA经心注射到了老化小鼠体内。A)先期剂量-效应研究结果表明，大于5.0μM剂量可显着降低衰老脑中·OH自由基的含量，同时脑铁随bs-5-YHEDA的浓度降低而降低。但当浓度超过20μM时，自由基清除效率变缓。B)心脏注射20μΜbs-5-YHDEA三周后，·OH自由基水平降至OD值0.13，bs-5-YHEDA给药时间越长，对自由基减少的影响越小。C)随着衰老，老年小鼠脑铁蛋白、转铁蛋白增加。而在用bs-5-YHEDA处理的衰老小鼠的大脑中，铁蛋白和转铁蛋白被逆转。D)在未治疗的老化小鼠的大脑中，坏死神经元分布较广。无LDLR结合段的5-YHEDA经心注射后预防神经元损伤作用不明显，大脑中的坏死神经元仍然与未治疗的老化脑中的神经元相差不多。但是，如果注射bs-5-YHEDA，老化小鼠脑中的神经元坏死减少(E，G)，因此，注射bs-5-YHEDA的老化脑中的血氧下降速度比未治疗的老化鼠或注射5-YHEDA的老化鼠快。注射bs-5-YHEDA的老化小鼠脑活跃区比另2组面积大82％。F，H)水迷宫测试表明，经bs-5-YHEDA处理的老化小鼠找到水台的时间比未处理的衰老和经5-YHEDA处理的衰老小鼠时间短。I)对铁和自由基检测表明bs-5-YHEDA大大延缓了衰老小鼠的脑铁和·OH自由基积累。*：p＜0.05。
具体实施方式
以下结合说明书附图对本发明做进一步详细说明，并给出具体实施方式。
本发明一种用于解除脑内神经元细胞铁胁迫的多肽，包括螯合铁序列和导肽序列，螯合铁序列、导肽序列均由氨酸残基组成，螯合铁序列包括若干个X序列，所述X序列由H、A、Y、E、D组成，所述H、A、Y、E、D任意排列，所述H为组氨酸残基，所述A为丙氨酸残基，所述Y为酪氨酸残基，所述E为谷氨酸残基，所述D为天冬氨酸残基；导肽序列为QSDIVAHAHLL，所述Q为谷氨酰胺残基简称、S为丝氨酸残基简称、I为异亮氨酸残基简称、V为缬氨酸残基简称、L为亮氨酸残基简称；导肽序列引螯合铁序列穿越血脑屏障解除神经细胞铁胁迫，且对其结合铁能力影响较小。
实施例
材料：5-YHEDA和bs-5-YHEDA肽由上海吉尔生物科技有限公司合成，人神经母细胞瘤细胞系SH-sy5y和人血管内皮细胞系HECV均购自美国标本组织培养库(ATCC，Manassas，VA)。昆明小鼠由温州医科大学动物中心提供。用58只经水迷宫检测表现出学习能力差(寻找水台时间>75s)和随后经免疫组化检测在胼胝体和海马区神经元坏死严重(>2个/mm2)的25月龄自然老化小鼠，观察5-YHEDA和bs-5-YHEDA对老化的治疗效果。
脑铁含量地形图扫描与测量：
取六只正常或SN小鼠(70mg/kg小鼠)用戊巴比妥生理盐水溶液麻醉处死后，取脑，用冷冻切片机切取冠状脑片(厚20mm)。称重后，将每个鼠脑的三个切片放在聚碳酸酯膜上，然后在X荧光工作站(中国北京高能物理研究所)下扫描脑切片各区域的铁含量。同一脑的剩余部分保留用于TUNEL或免疫胶体金检测。
另取六只正常或SN小鼠检测脑和脑脊液(CSF)内的铁含量。除细胞培养基外，所有样品均用电感耦合等离子体发射光谱仪(ICP,PerkinElmer Elan600,Fremont,CA)测量。测量之前，将样品分别冷冻干燥、称重和硝化。借助立体定位仪从受试小鼠第四脑室距离脑前窗PA-1.5mm,侧面1.5mm,腹面2mm硬脑膜处抽取脑脊液。用梯度浓度的FeCl3溶液定标。
5-YHEDA对铁的螯合与5-YHEDA聚团透射电镜观察
将合成的5-YHEDA粉末溶于蒸馏水(1mg/mL)中，分成两等份。一份用作对照，不加铁。在20μl第二份5-YHEDA样品中，加入将5μL0.01mM FeCl3溶液，37℃孵育3h。之后将每个样品滴在包被碳膜的铜网上，用1％(w/v)磷钨酸染色2分钟。风干后在配有EMAX-X射线能谱仪(日本，京都，Horriba)的透射电镜(H7650，日立，京都)下检测铁的分布。
等温释热滴定分析：为了确认铁原子与5-YHEDA之间的亲和性，先将纯的5-YHEDA寡肽用等温滴定量热(ITC)缓冲液作了彻底透析，然后将5-YHEDA寡肽稀释至200mM，并将FeCl3溶液稀释至1000mM。用VP-ITC微量计热器(MicroCal，GE Healthcare)测量铁与5-YHEDA寡聚物之间的结合。从结合热中减去配体稀释热，做成结合热曲线，用Origin软件中的单点结合模型该曲线拟合，用结合系数(Ka)，焓(ΔH)和熵(ΔS)确定相互作用程度。用Ka计算Kd(Kd＝1/Ka)。
红外光谱分析：为了确定5-YHEDA如何结合铁离子，将干燥的5-YHEDA:FeCl3反应物与K2Br粉末(1:5)混合，然后用红外光谱仪(NEXUS870，NICOLET)测量样品的红外(IR)光谱三次。纯5-YHEDA和K2Br用作对照。
质谱(图2.A)显示，具有五个H的肽段出现在m/z峰715.85处，与一重“YHEDA”肽的分子量(MW)相当。峰630.11是“YHED”寡肽，为YHEDA失去丙氨酸(MW 589)和几个氢原子后形成的片段。四H碎片和三H碎片分别位于894.81m/z和1192.40m/z。它们是“YHEDA-Y”和“YHEDA-YHE”寡肽(Y，H，E和D的MW分别为181、155、147和133)。因此，整个合成的肽的分子量为3574.5，为五重“YHEDA”。有关该肽的更多信息可从散布在以上峰附近的弱峰中分析获得。经蛋白质测序仪(PPSQ-21A/23A，Shimadzu Corp，Japan)，测得整个肽的氨基酸全序列为“YHEDA YHEDA YHEDA YHEDA YHEDA”，含LDLR结合片段的完整肽全序列为“YHEDA YHEDAYHEDA YHEDAYHEDA-
”。灰色涂抹部分为LDLR结合序列，合成的肽完全符合我们的设计。
细胞培养：SH-sy5y细胞用DMEM培养基于5％CO2下37℃培养12h，然后分铁胁迫组，铁+5-YHEDA组和铁+bs-5-YHEDA组(每组三盘细胞)。在铁胁迫组中，添加0.01mM FeCl3模拟AD脑CSF中铁的水平，其余两组添加0.01mM FeCl3，分别添加1.5μM 5-YHEDA或bs-5-YHEDA于培养液中(肽的剂量-效应预先做了检测)。将含或不含5-YHEDA或bs-5-YHEDA的细胞作对照，将所有细胞培养12h,然后将每个培养皿中的细胞一部分用2.5％戊二醛固定，用于扫描电镜检测。其余细胞用流式细胞仪进行凋亡分析(BD FACSCalibur，Franklin Lake)。分别收集每个培养皿中的培养基检测其中自由基和铁含量。
在相同条件下培养人血管内皮细胞(HECV)，作LDLR:bs-5-YHEDA免疫共沉淀。
细胞凋亡测定：分别收集不同培养皿中的SH-sy5y细胞，并在黑暗室温下用Annexin-V-FITC和碘化丙啶(IP)染色10分钟。用FACScan流式细胞仪(Becton Dickinsonand Company)分析细胞凋亡。用CellQuest Pro软件(Becton Dickinson Company)确定凋亡细胞占细胞总数的百分比。
羟自由基检测：将每个小鼠的CSF(每个50μL)以及各培养皿同体积的细胞培养基分别加入50μL 1％水杨酸溶液(w/v)。37℃振荡温育15分钟后，用酶标仪(SpectraMax M5，Molecular Devices)检测510nm波长下的透射率。以光密度值(OD)计量每个样本中·OH自由基水平。
抗5-YHEDA免疫血清制备：将100μL 5-YHEDA-生理盐水(0.1mg/mL)溶液与100μL不完全弗氏佐剂混合，每周经皮下或腹腔注射正常小鼠。2个月后，提取每只小鼠的全血，分离血清存在-20℃下备用。
免疫共沉淀：HECV细胞在标准条件下培养。当密度达到5×106个细胞/皿，将细胞用预冷的磷酸盐缓冲盐水(PBS)洗3次，并用含有蛋白酶抑制剂(PMSF)的100μL非变性裂解液在冰上裂解。将含bs-5-YHEDA或5-YHEDA肽(100μg/mL)的溶液与裂解液混合，然后将LDLR单抗加入蛋白质裂解物中，4℃孵育3小时。然后与蛋白A/G琼脂糖共孵育过夜，并通过离心回收纯化的蛋白复合物，再95℃加热5分钟。
蛋白质免疫印迹：配制10％的SDS-PAGE凝胶，电泳分离含等量蛋白质的各组样品(20μg)，然后将样品转移到硝酸纤维素膜上。室温下用TBST洗膜四次(每次5分钟)。再用1％牛血清白蛋白封闭膜，然后与抗体共孵育(用抗5-YHEDA血清检测LDLR和bs-5-YHEDA的共沉淀；用抗铁蛋白或抗转铁蛋白IgG于4℃孵育)过夜。然后用PBS洗涤五次，将膜与HRP标记的二抗室温孵育2h。最后用酶联化学发光法(Pierce Biotechnology，Inc，Chicago，IL)检测结合的抗体。GAPDH做内参。每个实验重复三遍。
剂量/持续时间-效应分析：为评估bs-5-YHEDA对脑保护的效果，我们做了剂量/持续时间-效应研究。在剂量-效应试验中，每周将200μL 0.0、0.5、1.0、5.0、20或40μΜbs-5-YHEDA生理盐水溶液通过心内注射到每只小鼠中。4周后，从每个受试小鼠脑室抽取50μLCSF。在持续时间-效应实验中，每周向每只小鼠心内注射200μL的20μM bs-5-YHEDA。从每只小鼠中提取50μL CSF，然后用水杨酸测量CSF中的·OH自由基含量。
放射自显影测定脑内bs-5-YHEDA的生物活性：心脏注射200μL20μM 3H同位素标记的bs-5-YHEDA，48h后，将正常小鼠的大脑冷冻切片(厚20μm)。在另一张载玻片滴上一系列梯度浓度3H同位素标记的bs-5-YHEDA溶液(用于标定3H-bs-5-YHEDA在脑中的生物活性)，冷冻干燥后与粘贴在载玻片上的脑切片一起放入装有3H感光胶片(Leica Inc，Deerfield，IL)的暗盒中，室温下曝光2周，然后将胶片显影、定影。将胶片上生成的图像每个像素位置的氚放射活性用来表示分布在大脑中的bs-5-YHEDA肽的生物活性。用免费软件NIHI(National Institutes of Health-Image,Wayne Rasband编写；可从zippy.nimh.nih.gov通过匿名FTP获得)测量图像的感光度曲线。获得的局部浓度以放射性单位(nCi/mg)表示。
5-YHEDA免疫组化染色：用0.1％牛血清白蛋白/PBS(w/v)封闭固定的脑切片。1h后，将抗5-YHEDA血清滴到切片上。4℃孵育过夜后洗涤。然后将HRP标记的羊抗小鼠IgG与切片一起孵育。2小时后再清洗，然后用DAB覆盖。30分钟后，用生物素染色。再用PBS洗3次，最后用显微镜观察。显示有棕色斑点的切片为5-YHEDA阳性，表明5-YHEDA到达大脑。
水迷宫测试：水迷宫用于评估小鼠的学习和记忆能力。水迷宫直径为1.5m，深度为0.6m，事先装有牛奶。在水槽中心，液面下1cm放一平台。实验时，表现出AD症状的SN昆明小鼠分未治疗组，5-YHEDA治疗组和bs-5-YHEDA治疗组。每周向后两组的每只小鼠心内注射200微升20μM 5-YHEDA或bs-5-YHEDA溶液。将6月龄的小鼠和未出现老化症状的老年小鼠用作对照。六周后，经1天适应期，所有小鼠均接受4天水迷宫测试。记录小鼠游泳找到平台的路径和所花费的时间，以评估个体的认知能力。
脑功能核磁共振成像分析：小鼠麻醉后，用核磁共振仪(E40，Flir Inc)在1.5T下观察小鼠脑中氧的代谢水平30分钟。再大脑中的信号亮度表征脑内血氧代谢水平。
血液学分析：为评估bs-5-YHEDA的副作用，根据国际临床协会参照区间和决策范围的混杂委员会协议[32]收集小鼠血液。将一毫升静脉血抽入装有EDTA-钾的真空管中进行血液学分析,检测白血球细胞计数，中性粒细胞百分比，淋巴细胞百分比，单核细胞百分比，嗜碱性粒细胞百分比，嗜酸性粒细胞百分比，红细胞计数，血细胞比容，平均红细胞体积，平均红细胞血红蛋白浓度，红细胞分布宽度，血小板计数和平均血小板体积。
临床生化分析：为确定bs-5-YHEDA对肾脏或肝脏的副作用，我们做了临床化学分析。具体地，使用SpectraMax微量滴定板读数器(Molecular Devices，LLC)和MaxDiscovery血液尿素氮酶试剂盒(Bioo Scientific Corporation)测定血液尿素氮。QuantichromCreatinine Assay Kit(BioAssay Systems)用于测量血清中的肌酐。用自动分析仪(Selectra Junior Spinlab 100，Vital Scientific，Dieren，荷兰)测定天冬氨酸转氨酶和丙氨酸转氨酶的血清活性。
胶体金免疫组织化学染色和铁：bs-5-YHEDA共定位扫描
为了确定铁是否在体内与bs-5-YHEDA肽结合，我们做了胶体金免疫组织化学扫描电镜分析。将脑切片用抗5-YHEDA小鼠血清免疫组化染色，清洗后立即与胶体金标记的IgG溶液孵育。在室温下2小时后，清洗样品并用叔丁醇冷冻干燥。用金颗粒真空溅射后，在SEM(S-3000N，日立，Kyoto)下观察。用自带X射线能谱(EX-450，HORIBA EMAX，日本)仪的SEM扫描每个切片中的铁分布，同时获得铁：bs-5-YHEDA共定位形貌。
TUNEL分析：用TUNEL试剂盒(上海Sangon Biotech Inc，中国)检测大脑中的细胞坏死。将冷冻的小鼠脑切片在HEPES(Na-N-2-羟乙基哌嗪-N-乙烷磺酸)溶液中孵育1小时，然后用10mM H2O2和20mM孕酮处理。用PBS洗涤然后用0.1％Triton-X 100通透，在黑暗中37℃进行TUNEL温育1小时。随后将样品的一部分用DAB染色。最后在显微镜观察。亮点或褐色斑点表示组织中的坏死细胞。
结果
如图1所示SN小鼠大脑中的铁和羟基自由基含量较高，且神经元坏死严重：SN小鼠脑内的铁比正常小鼠的高，平均每克脑含0.72μg铁，而正常小鼠每克脑仅铁0.43μg铁(图1.A)。铁含量地貌图(图1.A)表明：在AD脑内铁高度集中在胼胝体、皮质、海马、视前外侧区、扣带状皮质和杏仁核(图1.A-a，c，d，e，g，i，j)。在脑室，前肱前区和外侧内嗅皮层较少(图1.A-b，h)。相比之下，除了脑室(图1A-b)和海马CA1(与图1.A-b相邻)有零星密集外，正常鼠脑的大部分区域铁的水平相对较低。与之对应，在SN脑中，·OH自由基水平比正常脑(图1.B)高140％，坏死神经元(图1.C-a中的棕色点)分布较广。
图2所示，质谱(图2.A)显示，具有五个H的肽段出现在m/z峰715.85处，与一重“YHEDA”肽的分子量(MW)相当。峰630.11是“YHED”寡肽，为YHEDA失去丙氨酸(MW 589)和几个氢原子后形成的片段。四H碎片和三H碎片分别位于894.81m/z和1192.40m/z。它们是“YHEDA-Y”和“YHEDA-YHE”寡肽(Y，H，E和D的MW分别为181、155、147和133)。因此，整个合成的肽的分子量为3574.5，为五重“YHEDA”。有关该肽的更多信息可从散布在以上峰附近的弱峰中分析获得。经蛋白质测序仪(PPSQ-21A/23A，Shimadzu Corp，Japan)，测得整个肽的氨基酸全序列为“YHEDA YHEDA YHEDA YHEDAYHEDA”，含LDLR结合片段的完整肽全序列为“YHEDA YHEDA YHEDAYHEDA YHEDA-
”。灰色涂抹部分为LDLR结合序列，合成的肽完全符合我们的设计。
5-YHEDA肽长度约为150nm(图2.B-c)，呈线状，但与FeCl3孵育后卷曲并聚结(图2.B-d)；铁(图2.B-d右图中的亮点)沿5-YHEDA同向分布，说明与FeCl3共孵育后，铁原子与肽之间发生了结合。ITC释热分析证实了这种结合。如图2.C-b所示，在FeCl3滴定后，5-YHEDA的焓降低了91255kcal/mol，Gibbs能降低了8973kcal/mol。与之对比，用ITC缓冲液滴定5-YHEDA时，几乎没有热量释放(图2.C-a，每次滴定释放0.26kcal/mol或更低)。红外色谱深入分析表明5-YHEDA肽在残基His，Tyr，Asp和Glu处结合铁。与FeCl3孵育后，代表Glu和Asp羧基中C＝O基团振动的1600-1300cm-1谱带中的峰发生了变化。720cm-1处的峰(代表Tyr的酚基)减弱(图2.D-b)。2375cm-1处的吸收风顶点代表C-N键的伸展；与FeCl3孵育后，代表His残基咪唑环中C＝N键伸展的1700-1615cm-1谱带的峰均减弱(图2.D-b)。这些结果表明在这些基团存在被铁原子螯合或影响的可能性。5-YHEDA和bs-5-YHEDA可通过减少自由基保护细胞
铁离子可催生自由基。如果发生在大脑中，神经元将受到损害，从而导致神经数量减少和认知障碍。为了确认5-YHEDA或bs-5-YHEDA可保护神经元免受铁催生的自由基损害，我们将合成的5-YHEDA或bs-5-YHEDA处理了铁胁迫(含15μM FeCl3，模拟AD CSF)培养的神经母细胞瘤细胞SH-sy5y。在处理之前，我们测量了YHEDA重复序列在铁结合和对·OH自由基还原的效率，以获得最佳的肽长度和合适剂量。如图3.A所示，随着YHEDA的重复数的增加，螯合铁和清除·OH的效率增加，直到长度增加到四重复，其作用减慢。因此，我们向培养基中添加了5个重复的YHEDA或bs-5-YHEDA肽，观察到培养基中自由基的数量随肽浓度升高而降低(图3.B)。
5-YHEDA或bs-5-YHEDA两种肽都可以保护富铁培养基中细胞(图3.C–E)。分光光度法和ICP等离子体分析表明，36h后，培养基中的·OH自由基从0.34降至0.19，游离铁离子减少了三分之一(图3.E)。特别是加入1.5μM 5-YHEDA或bs-5-YHEDA中，在铁胁迫培养的SH-sy5y细胞得以存活，它们的轴突和树突完好无损，树突上的棘突丰富(如图3.C-d中的箭头所示)，细胞表现出活跃的胞质运输，可观察到沿着轴突分布有运输囊泡(图3C-d中的箭头所示)。相反，如果没有5-YHEDA或bs-5-YHEDA，富铁培养基中的细胞萎缩，它们的轴突和树突收缩，树突棘的数量减少(图3.C-b)。看不到运输囊泡(图3.C-b)。说明铁胁迫会损害细胞的生理活性，但是5-YHEDA或bs-5-YHEDA可使受胁迫的细胞脱离自由基的危险环境。
免疫共沉淀表明，bs-5-YHEDA可结合LDLR。如图4.A泳道3所示，在LDLR上方，分子量约为100kDa处有一较粗的黑带，该黑带约为bs-5-YHEDA和LDLR的总分子量，说明bs-5-YHEDA与LDLR发生了结合而共沉淀。相反，当LDLR结合段缺失时，将5-YHEDA与LDLR混合，混合物电泳出两条细条带：一条与LDLR处于同一水平，另一条跑到底部，每个条带附近都有些淡而模糊的条带(可能是其他可与LDLR或5-YHEDA结合的痕量分子)。在泳道5的100kDa处未观察到比泳道3同一位置更深的条带，这表明缺少LDLR结合片段的5-YHEDA不会与LDLR相互作用。图4.B-c显示连接了LDLR结合段的治疗性5-YHEDA肽注入心脏后用免疫组化染为棕色散布在脑切片中。相反，经心注射了未连接LDLR结合段的5-YHEDA，小鼠的脑切片未观察到免疫染色(图4.B-b)。3H同位素放射自显影进一步证明，在心脏注射200μL 20μmM 3H-bs-5-YHEDA 6天后，脑脊液中剩余的3H-bs-5-YHEDA浓度约为0.26μM，仍为活跃浓度(图4.C-b)。胶体金免疫检测进一步证明，在SN小鼠大脑中，经心注射到达脑内的bs-5-YHEDA肽结合了铁。结果bs-5-YHEDA附近的铁聚集在肽处与肽共定位，造成肽附近区域铁浓度降低(图4.D-c)。相反，注入未与LDLR结合段相连的5-YHEDA，大脑中未观察到金颗粒，并且铁背景更多(图4.D-b)。因此，没有LDLR结合片段的5-YHEDA很难穿过BBB入脑螯合除铁。
bs-5-YHEDA可保护大脑并改善SN小鼠的认知能力：
剂量-效应和持续时间-效应研究表明，随着浓度的增加，脑脊液中的铁和·OH自由基减少。当浓度增加至5μM时，每周经心注射200μL bs-5-YHEDA，4周后，CSF的·OH自由基光密度降至0.12；同时CSF的铁浓度从18μM降低至12μM(图5.A)。每周注射200μL20μM bs-5-YHEDA，三次后，CSF中的·OH自由基的OD降至0.13，CSF铁降低至13μM；四周后，自由基的下降变缓(图5.B)。在正式实验中，我们每次用200μL的20μM bs-5-YHEDA处理小鼠。从图5.C可看出，随着年龄的增长，在6个月大以后，SN小鼠中的脑铁储藏蛋白-铁蛋白平均增加了60％，铁内运蛋白转铁蛋白平均增加了30％。然而，当用bs-5-YHEDA处理SN小鼠约6周时，脑铁蛋白和转铁蛋白的增加被逆转，这表明bs-5-YHEDA可通过减少铁积聚和减少铁向脑内运输防止脑铁水平升高。结果，SN大脑中的神经元比未治疗的SN小鼠(图.5D)坏死少(图5D中箭头所指的亮点是坏死的神经元)，保持较好的组织性，即bs-5-YHEDA规避了铁催生的自由基攻击保护了神经元。相比之下，用无LDLR结合段的5-YHEDA处理的小鼠，其大脑中未检测到上述现象，脑内铁和自由基的水平几乎与未治疗的SN小鼠相同(图.5-I)。
此外，我们还观察到，在bs-5-YHEDA处理之后，脑血氧代谢水平(图5.E中所示的大脑明亮区域)比未处理的SN小鼠高出82％。水迷宫(图5.F和H)显示，训练后，未经处理的SN小鼠和用5-YHEDA处理的SN小鼠比经bs-5-YHEDA处理的SN小鼠笨拙。bs-5-YHEDA处理的SN小鼠平均只花费57秒，游220cm即找到水台，比未处理的SN小鼠和经5-YHEDA处理的SN快近25秒，少90cm(图5F和H)。这表明合成的bs-5-YHEDA肽延缓了小鼠的认知和记忆力下降。没有结合LDLR片段，5-YHEDA肽很难入脑，更不用说清除脑中的铁和自由基了。
bs-5-YHEDA未对肝脏，肾脏和血液产生副作用，反而可缓解衰老引起的炎症和贫血
好药不仅应有高的疗效，而且还不应对人体造成副作用。为了确定bs-5-YHEDA是否对机体有不利影响，我们对经bs-5-YHEDA治疗后的小鼠作了肝、肾功能和血常规检测。如表1所示，在衰老过程中，丙氨酸氨基转移酶和天冬氨酸氨基转移酶的水平增加了近两倍，尽管bs-5-YHEDA没有逆转衰老引起的肝损伤，但也未加剧其恶化。bs-5-YHEDA和衰老均未对肾脏产生明显不良影响。SN小鼠和bs-5-YHEDA治疗的小鼠的血肌酐和血尿氮水平与对照组的肌酐几乎相同，这表明bs-5-YHEDA不会损害肾脏。
衰老和bs-5-YHEDA对平均红细胞体积或中性粒细胞百分比的影响很小，bs-5-YHEDA对衰老相关的红细胞分布宽度和单核细胞百分比的增加几乎没有改变。但是，bs-5-YHEDA可逆转衰老造成的红细胞计数、血细胞比容、血小板计数和平均红细胞血红蛋白浓度的下降。同样，给SN小鼠施用bs-5-YHEDA后，老化造成的淋巴细胞百分比、平均血小板体积和嗜酸性粒细胞百分比的增加被逆转，表明bs-5-YHEDA具有抗炎作用，可以部分缓解贫血。
表.1 临床生化与血常规检测结果
注解：AST：天冬氨酸转氨酶，ALT：谷氨酸-丙酮酸转氨酶，RR：参考范围；SCr：血清肌酐，BUN：血液尿素氮；RBC：红细胞计数，MCV：平均红细胞体积，RDW：红细胞分布宽度，HCT：血细胞比容，MCH：平均红细胞血红蛋白，MCHC：平均红细胞血红蛋白浓度，WBC：白细胞计数，NE：中性粒细胞百分比，EO：嗜酸性粒细胞百分比，BA：嗜碱性粒细胞百分比，LY：淋巴细胞百分比，MO：单核细胞百分比，PLT：血小板计数，MPV：平均血小板体积。↑：与普通鼠标相比；▲：与衰老的老鼠相比。p＜5％。
Claims (3)
1.一种用于解除脑内神经元细胞铁胁迫的多肽，其特征在于包括螯合铁序列和导肽序列，所述螯合铁序列、导肽序列均由氨酸残基组成，所述导肽序列为QSDIVAHAHLL，所述Q为谷氨酰胺残基简称、S为丝氨酸残基简称、I为异亮氨酸残基简称、V为缬氨酸残基简称、L为亮氨酸残基简称。
2.如权利要求1所述的一种用于解除脑内神经元细胞铁胁迫的多肽，其特征在于所述螯合铁序列包括若干个X序列，所述X序列由H、A、Y、E、D组成，所述H、A、Y、E、D任意排列，所述H为组氨酸残基，所述A为丙氨酸残基，所述Y为酪氨酸残基，所述E为谷氨酸残基，所述D为天冬氨酸残基。
3.如权利要求1所述的一种用于解除脑内神经元细胞铁胁迫的多肽，其特征在于所述导肽序列引螯合铁序列穿越血脑屏障解除神经细胞铁胁迫。
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