CN106822864A - 一种抑制前蛋白转化酶枯草溶菌素9表达的方法 - Google Patents
一种抑制前蛋白转化酶枯草溶菌素9表达的方法 Download PDFInfo
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- CN106822864A CN106822864A CN201710106759.1A CN201710106759A CN106822864A CN 106822864 A CN106822864 A CN 106822864A CN 201710106759 A CN201710106759 A CN 201710106759A CN 106822864 A CN106822864 A CN 106822864A
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- lunasine
- lunasin
- proprotein convertases
- subtilisin
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Abstract
本发明公开了一种抑制前蛋白转化酶枯草溶菌素9表达的方法，通过使用露那辛抑制机体前蛋白转化酶枯草溶菌素9(PCSK9)的产生，降低前蛋白转化酶枯草溶菌素9的血清水平。本发明还公开了一种露那辛的新用途，可作为前蛋白转化酶枯草溶菌素9抑制剂在治疗前蛋白转化酶枯草溶菌素9介导的相关疾病，包括高胆固醇血症、肥胖、糖尿病、肾病等。本发明首次发现露那辛对前蛋白转化酶枯草溶菌素9的抑制作用，开辟了露那辛新的应用领域，具有极高的市场应用价值。
Description
技术领域
本发明属于生物医药领域，具体涉及一种抑制前蛋白转化酶枯草溶菌素9(PCSK9)表达的方法，可通过使用露那辛(lunasin)抑制机体前蛋白转化酶枯草溶菌素9的产生，降低前蛋白转化酶枯草溶菌素9的血清水平。可进一步将露那辛作为前蛋白转化酶枯草溶菌素9抑制剂用于制备治疗前蛋白转化酶枯草溶菌素9介导的相关疾病的药物。
背景技术
前蛋白转化酶枯草溶菌素9(PCSK9)(proprotein convertase subtilisin/kexin9,PCSK9)是一种由692个氨基酸组成的糖蛋白，属前蛋白转化酶(proproteinconvertases,PCs)家族中的第九个成员，是一种分泌型丝氨酸蛋白酶，主要在肝脏和肠道等组织中表达，然后分泌到血液中(J Biol Chem.2004；279(47):48865-48875；TrendsBiochem Sci.2007；32(2):71-77)。PCSK9进入血液循环后可与肝细胞表面的低密度脂蛋白受体(low density lipoprotein receptor,LDL-R)的表皮生长因子样结构域特异性结合，引导其进入肝细胞到达溶酶体，使LDL-R在溶酶体中降解，从而导致肝细胞表面LDL-R减少，进而降低肝脏结合和清除LDL-C的能力，最终导致血液中LDL-C水平升高(Proc Natl AcadSci U S A.2008；105(35):13045-13050)。因此，可通过抑制PCSK9治疗高胆固醇血症(AnnuRev Pharmacol Toxicol.2014；54:273-293)。此外，最新研究显示，PCSK9升高与肥胖及2型糖尿病(Pediatr Diabetes.2017.PMID:28093849,DOI:10.1111/pedi.12490；DiabetesMetab Res Rev.2016；32(2):193-199.)密切相关，也与肾病综合症及蛋白尿等慢性肾病(Int Urol Nephrol.2017.PMID:28084558，DOI:10.1007/s11255-017-1505-2)密切相关，因此，通过抑制PCSK9可以成为预防和治疗这些与其相关的疾病的重要手段。
目前已有多种PCSK9抑制剂处于研发阶段或已获批上市成为治疗高胆固醇血症的药物，其中主要包括：1、抗PCSK9抗体抑制剂，如美国Amgen公司的Evolocumab(AMG145)及Regeneron/Sanofi公司的Alirocumab(REGN727/SAR236553)已在美国和欧洲获批上市(JClin Pharmacol.2017；57(1):7-32)；2、小干扰RNA(SiRNA)抑制剂，如Alnylam公司的ALN-PCS可抑制PCSK9的转录，目前已进入I期临床试验(Pharmacol Ther.2016；164:183-194)；3、小分子化合物，如小檗碱可以抑制PCSK9的转录和翻译(J Biol Chem.2015；290(7):4047-4058；Atherosclerosis.2008；201(2):266-273)。
露那辛(lunasin)是一种最初从大豆中分离得到的活性肽，由43个氨基酸残基组成，其序列为:SKWQHQQDSCRKQLQGVNLTPCEKHIMEKIQGRGDDDDDDDDD(SEQ ID NO:1)，分子量为5KDa(J Biol Chem.1987；262(22):10502-10505)。已有研究表明，多肽露那辛具有抗肿瘤活性，在体外可抑制非小细胞肺癌细胞的增殖(Oncotarget.2015；6(7):4649-4662)，抑制乳腺癌细胞的迁移和侵袭(Oncol Rep.2016；36(1):253-262)，在体内可抑制小鼠皮肤癌和乳腺癌的发生(J Food Sci.2010；75(9):H311-316)。
发明内容
本发明的目的在于提供一种抑制PCSK9的方法，具体说是通过用多肽露那辛(lunasin)抑制肝细胞组织中前蛋白转化酶枯草溶菌素9(PCSK9)表达的方法。PCSK9是一种分泌型丝氨酸蛋白酶，主要在肝脏、小肠等组织中表达后分泌到血液中，现已发现PCSK9与高胆固醇血症、肥胖、2型糖尿病、肾病综合症及蛋白尿等慢性肾病密切相关，本发明提供了一种采用多肽露那辛有效抑制PCSK9生成的方法，可应用于制备上述与PCSK9相关的疾病的制剂或药物。
本发明具体技术方案如下：
一种抑制前蛋白转化酶枯草溶菌素9表达的方法，使用露那辛抑制机体前蛋白转化酶枯草溶菌素9的产生，降低前蛋白转化酶枯草溶菌素9的血清水平，所述露那辛的氨基酸序列如SEQ ID No：1所示。
所述露那辛氨基酸C末端可以是非酰胺化或酰胺化的。
所述露那辛可通过基因工程方法、化合合成方法或者从天然植物材料中提取的方法制备得到。
上述方法，可将露那辛与其它前蛋白转化酶枯草溶菌素9抑制剂联合使用，所述前蛋白转化酶枯草溶菌素9抑制剂包括PCSK9单克隆抗体、PCSK9小干扰RNA、PCSK9基因沉默寡核苷酸、小檗碱及其衍生物、姜黄素、海藻寡糖中的一种或几种。
本发明还公开了一种露那辛的新用途，其特征在于露那辛作为前蛋白转化酶枯草溶菌素9抑制剂用于制备治疗前蛋白转化酶枯草溶菌素9介导的相关疾病的药物，包括高胆固醇血症、肥胖、糖尿病(2型糖尿病)、肾病(肾病综合症及蛋白尿等慢性肾病)。
上述用途，可将露那辛可制备成药学上接受的任何一种制剂，优选片剂、胶囊、滴剂、粉针剂或水针剂。
上述药物包括作为活性成分的露那辛及可药用载体。较佳的，本发明的药物有0.1-99.9％重量百分比的作为活性成分的露那辛。
“可药用载体”包括但不限于：离子交换材料、氧化铝、硬脂酸铝、卵磷脂、自乳化药物传递系统(SEDDS)如d-维生素E聚乙二醇1000琥珀酸酯、吐温或其他类似聚合介质等药物制剂用的表面活性剂、血清蛋白如人血清白蛋白、缓冲物质如磷酸盐、氨基乙酸、山梨酸、山梨酸钾、饱和植物脂肪酸部分甘油酯混合、水、盐、电解质如硫酸盐精蛋白、磷酸氢二钠、磷酸氢钾、氯化钠、锌盐、硅胶、硅酸镁等。聚乙烯吡咯酮、纤维素物质、聚乙烯醇、羧甲基纤维素钠、聚丙烯酸酯、乙烯-聚氧乙烯-嵌段聚合物和羊毛脂、环糊精如α-、β-、γ-环糊精或其经化学修饰的衍生物如2-和3-羟丙基-β-环糊精等羟烷基环糊精或其他可溶性衍生物等均可用于促进露那辛的药物传递。
其他可药用辅料如填充剂(如无水乳糖、淀粉、乳糖珠粒和葡萄糖)、粘合剂(如微晶纤维素)、崩解剂(如交联羧甲基淀粉钠、交联羧甲基纤维素钠、低取代羟丙基纤维素和交联PVP)、润滑剂(如硬脂酸镁)、吸收促进剂、香味剂、甜味剂、稀释剂、赋形剂、润湿剂、溶剂、增溶剂和着色剂等也可加入本发明的药物中。
上述药物的治疗有效量为0.001-100mg/kg/d之间，可用于相关疾病的单一用药或联合用药治疗，为本领域技术人员能够理解的范围。
附图说明
图1：lunasin呈剂量及时间依赖性抑制HepG2细胞中PCSK9的mRNA表达水平。图1-1：不同浓度lunasin对PCSK9mRNA表达的影响；图1-2：lunasin不同作用时间对PCSK9mRNA表达的影响。*，**，***及****表示与正常组比较P<0.05，P<0.01，P<0.001及P<0.0001(n＝3,means±SEM)。
图2：lunasin呈剂量及时间依赖性抑制HepG2细胞PCSK9蛋白的表达。图2-1：不同浓度lunasin对HepG2细胞内PCSK9蛋白表达的影响；图2-2：不同浓度lunasin对HepG2细胞分泌PCSK9蛋白的影响；图2-3：lunasin不同作用时间对HepG2细胞内PCSK9蛋白表达的影响。*及**表示与正常组比较P<0.05及P<0.01(n＝3,means±SEM)。
图3：Western blot检测PCSK9蛋白在ApoE-/-小鼠肝脏内的表达。####表示与正常对照组比较，P<0.0001；***表示与模型组比较，P<0.001(n＝8,means±SEM)。
图4：免疫荧光检测lunasin对ApoE-/-小鼠肝脏分泌PCSK9蛋白的影响。
具体实施方式
以下通过实施例说明本发明的具体步骤，但不受实施例限制。
本发明中所使用的术语，除非另有说明，一般具有本领域普通技术人员通常理解的含义。
下面结合具体实施例并参照数据进一步详细描述本发明。应理解，该实施例只是为了举例说明本发明，而非以任何方式限制本发明的范围。
在以下实施例中，未详细描述的各种过程和方法是本领域中公知的常规方法。
下述实例中所用的材料、试剂等，如无特殊说明，均可从商业途径获得。以下通过实施例进一步说明本发明：
实施例1 lunasin有效抑制HepG2细胞内PCSK9基因的表达
多肽露那辛(lunasin)药物采用本发明实验室建立的基因工程方法(Setrerrahmane,S.Appl Biochem Biotechnol 174(2014),612-622)制备得到。人肝癌细胞株HepG2(购自中科院上海细胞库)用含10％FBS(Cat:10099141,Gibco)MEM培养基(41500034,Gibco)重悬后，以5×105个细胞/孔接种于6孔板，置于37℃5％CO2培养箱中培养汇合至80％时，弃原培养基，PBS洗一次，每孔加入1ml Opti-MEM培养基(Cat:51985042,Gibco)平衡1h后加药处理。量效实验给药方法：lunasin给药浓度为0、0.2、1及5μM，给药时间为24h；时效性实验给药方法：lunasin给药浓度为5μM，给药时间为1、2、4、8、16及24h。药物处理结束后，采用Quantitative Real Time-PCR(qRT-PCR)检测lunasin对HepG2细胞内PCSK9基因表达水平的影响。
1.总RNA提取
细胞经药物处理后，6孔板每孔加入1ml RNAiso Plus(Takara,9108Q)，反复吹打至无明显沉淀。于室温静置5min，加入200μl氯仿，剧烈振荡15s，待溶液充分乳化，室温静置5min后，12000rpm，4℃离心15min。小心吸取400μl上清液，注意不要触碰到蛋白带和EP管管壁。于上清中加入异丙醇(等体积)，上下颠倒混匀，室温静置10min后，4℃，12000rpm，离心10min，获得RNA沉淀。缓慢加入75％的乙醇，轻轻上下颠倒清洗5-6次，7500rpm，4℃离心5min后，尽除乙醇。室温干燥沉淀约10min，至无液体残余，加入适量RNase-free水溶解后-80℃保存备用。用NanoDrop 2000/2000c分光光度计测定各样品的RNA浓度和纯度。RNA尽量于当天逆转录成cDNA-20℃储存。
2.cDNA合成
按表1依次加入各试剂以去除RNA中的基因组DNA。然后按表2建立逆转录体系合成cDNA，每20μl反应体系中加入1μg RNA。相关试剂购自大连宝生物Takara公司。
表1 去除基因组DNA反应体系
混匀后，瞬时离心，42℃2min(或者室温5min)，4℃hold。
表2 cDNA合成反应体系
混匀后，瞬时离心，进行cDNA的合成。逆转录条件如下：37℃15min，85℃5s,4℃保存。
3.qRT-PCR检测
qRT-PCR引物序列见表3，由上海捷瑞生物工程有限公司合成。按表4建立反应体系，试剂由大连宝生物Takara公司提供(Cat#RR820A)，混匀后，瞬时离心，并按表5所示程序进行Realtime PCR反应。根据以下公式绘制各基因PCR扩增效率曲线：E(％)＝(10-/slope-1)×100。取样品100ng cDNA如上进行Realtime PCR反应，同时设置NTC组(the no-temple control)，用ΔΔCt法分析实验结果。其公式为：2-ΔΔCT＝2-[(CT gene of interest-CT internal control)sample A -(CT gene of interest–CT internal control)sample B]
表3 qRT-PCR引物表
表4 20μl PCR反应体系
表5 PCR反应程序
qRT-PCR法检测lunasin对HepG2细胞内PCSK9基因表达水平的影响，结果见图1。量效实验结果显示：0.2、1、5μΜlunasin作用于HepG2细胞24h后，与正常组比较，lunasin呈剂量依赖性抑制HepG2细胞内PCSK9基因的表达，其中，5μΜlunasin作用最显著(P<0.0001)(图1-1)。时效实验结果显示：5μΜlunasin处理HepG2细胞1、2、4、8、16、24h后，lunasin呈剂量依赖性抑制HepG2细胞内PCSK9基因的表达，其中，5μΜlunasin作用24h抑制作用最显著(P<0.0001)(图1-2)。
由此证明，lunasin在转录水平呈剂量和时间依赖性抑制HepG2细胞PCSK9基因的表达，5μΜlunasin作用于HepG2细胞24h，抑制效应最为显著。
实施例2 lunasin有效抑制HepG2细胞PCSK9蛋白表达
1.细胞总蛋白提取
培养于6孔板内的HepG2细胞经药物分组处理后，收集1ml细胞培养液上清以10kd超滤管浓缩并定容至100μl。同时HepG2细胞以4℃预冷的PBS洗涤3次，每1×106个细胞中，加入100μl RIPA细胞裂解液(PMSF:RIPA＝1:100)，用细胞刮将贴壁细胞轻轻刮下，冰上裂解30min后，冰浴条件下超声处理(超声模式为超声1s，间歇2s，总时长1min)，4℃，12000rpm离心10min，收集细胞裂解液上清，并用BCA法测定各组蛋白含量。根据各组蛋白含量，稀释各样品至同一浓度，以确保蛋白上样量一致，加入5×Loading Buffer，混匀后煮沸5min，使蛋白变性，-20℃保存备用。
2.SDS-PAGE电泳
按表6制备12％SDS-PAGE胶，每孔上样量为20～30μg蛋白，对照孔上5μl预染Marker,对样品进行电泳分离，电泳条件为：80V电泳约30min，120V继续电泳至溴酚兰到红线以下。
表6 SDS-PAGE胶配制表
3.转膜
电泳结束后，依据分离胶的大小，剪取0.22μm PVDF膜1张和滤纸6张。PVDF膜先用甲醇浸泡5min，双蒸水中漂洗2min，再将PVDF膜和滤纸均于转膜缓冲液中浸泡10min后，直接在装置的负极板上制作“三明治”，从负至正依次为：多孔性垫片-3层滤纸-胶-PVDF膜-3层滤纸-多孔性垫片。注意四边对齐并小心赶出气泡，冰水浴根据目的蛋白分子量60V-100V恒压转膜100min。转膜结束后，取出PVDF膜标记正反面后，TBST清洗5min，加入丽春红染色3-5min至出现清晰条带，拍照记录。
4.封闭
TBST漂洗2-3次，每次5min，去除丽春红后，于10ml封闭液(含5％脱脂奶粉的TBST)中室温封闭1h。TBST洗3次，5min/次。
5.抗体孵育
根据目的蛋白分子量，将膜剪成相应大小，并剪去左上角作标记。用含5％脱脂奶粉的TBST以1：1000稀释抗PCSK9抗体(Cat:ab125251,abcam)，加至相应的PVDF膜上，4℃孵育过夜。TBST洗3次，5min/次。用二抗稀释液以1:5000稀释二抗，加至PVDF膜上，室温于水平摇床孵育2h。
6.显色鉴定
TBST清洗PVDF膜3次，5min/次。加ECL显色液于PVDF膜上，天能Tanon凝胶成像仪成像，分析实验结果。
Western blot法检测HepG2细胞内和分泌至细胞培养液上清中的PCSK9蛋白表达水平。结果(图2)显示，0.2、1、5μM lunasin作用于细胞24h后，Western blot法检测HepG2细胞内(图2-1)及分泌至细胞培养液上清中的PCSK9蛋白(图2-2)。由此说明，与正常组相比，lunasin均呈剂量依赖性抑制HepG2细胞内及分泌至细胞培养液中的PCSK9蛋白水平，且5μMlunasin抑制作用最显著(P<0.01)。进一步检测lunasin不同作用时间对HepG2细胞内PCSK9蛋白表达水平的影响，结果如图2-3所示，5μM lunasin作用于细胞1、2、4、8、16、24h后，lunasin呈时间依赖性抑制HepG2细胞内PCSK9蛋白表达，且24h抑制效应最显著(P<0.01)。由此证明，lunasin呈剂量和时间依赖性抑制HepG2细胞表达PCSK9蛋白。
因此，实例1和2实验结果证明，lunasin可在转录和翻译水平显著抑制HepG2细胞中PCSK9基因的表达。
实施例3 Lunasin腹腔给药可以有效抑制ApoE-/-小鼠肝脏组织PCSK9的表达
1.动物分组及药物处理
6周龄ApoE-/-小鼠及C57BL/6小鼠各16只均购自北京维通利华实验动物技术有限公司，许可证号:SCXK(京)2012-0001。动物喂养于SPF级动物房，维持室温25℃，湿度40％-70％，明暗各12h，小鼠自由进食饮水。小鼠给予基础饲料适应性喂养1周后，ApoE-/-小鼠给予高脂饲料(Research Diets,D12108C)，C57BL/6小鼠给予基础饲料，喂养6周后，ApoE-/-小鼠随机分为模型组(生理盐水)、给药组(0.5μmol/kg lunasin)，C57BL/6小鼠随机分为正常对照组(生理盐水)和阴性药物组(0.5μmol/kg lunasin),每组各8只动物。采用腹腔注射方式给药，给药剂量为0.5μmol/kg，1次/天，连续给药4周后，小鼠麻醉后处死，分离肝脏，进行后续实验。
2.Western blot检测lunasin对ApoE-/-小鼠肝脏PCSK9蛋白表达的影响
分离小鼠肝组织后，眼科剪剪碎，4℃预冷的生理盐水洗涤3次，以除去残血，加蛋白裂解液(铂优生物公司产品)(每100mg肝脏加入600μl裂解液)匀浆后，冰上裂解30min(隔10min涡旋振荡1次)，12000r 4℃离心15min，取上清至离心管中，-20℃保存备用。BCA法测蛋白浓度后采用Western blot方法检测小鼠肝脏PCSK9蛋白表达水平，具体方法同实施例2。
结果如图3所示，C57BL/6小鼠腹腔注射0.5μmol/kg lunasin 4周后，其肝组织内PCSK9蛋白水平与正常对照组相比无显著差异，说明lunasin不影响正常C57BL/6小鼠肝组织表达PCSK9蛋白。另外，与正常对照组比较，ApoE-/-模型组小鼠肝组织PCSK9蛋白水平显著升高，差异显著(P<0.0001)，然而ApoE-/-小鼠腹腔注射0.5μmol/kg lunasin 4周，则可显著抑制其肝组织内PCSK9蛋白表达水平升高(P<0.001)。由此可见，lunasin不影响正常小鼠肝组织分泌PCSK9蛋白，然而可以显著抑制ApoE-/-小鼠PCSK9蛋白水平升高。
3.免疫荧光检测lunasin对ApoE-/-小鼠肝脏分泌PCSK9蛋白水平的影响
小鼠肝组织分离后，立即放入4％多聚甲醛中固定48h后，常规脱水、透明、浸蜡、包埋、切片，切片厚度4μm。切片常规脱蜡复水后，进行免疫荧光学检测。
(1)组织切片后，60℃烤1h；
(2)依次脱蜡：二甲苯Ⅰ15min,二甲苯Ⅱ15min，无水乙醇5min，95％乙醇5min，85％乙醇5min，75％乙醇5min，PBS 5min；
(3)脱蜡后的切片置于柠檬酸缓冲液中，加热煮沸20min，自然冷却后，PBS洗3次，5min/次；
(4)3％H2O2室温10min，PBS洗3次，5min/次；
(5)10％山羊血清封闭，室温30min；
(6)弃封闭液，滴加PCSK9一抗(1：200)，4℃孵育过夜；
(7)37℃复温45min，PBS洗3次，5min/次；
(8)滴加Alexa Fluor 488标记的羊抗兔二抗(1：500)，37℃避光孵育60min；
(9)PBS洗3次，5min/次；
(10)滴加DAPI,染色10min，晾干，加甘油封片。倒置荧光显微镜下观察拍照。
结果(图4)显示：正常对照组C57BL/6小鼠和阴性药物组C57BL/6小鼠肝组织间隙可见少量绿色荧光，表示该小鼠肝组织内有少量PCSK9蛋白分泌；模型组ApoE-/-小鼠肝组织间隙有大量明亮的绿色荧光，表示其肝组织分泌PCSK9蛋白明显增多；给予lunasin腹腔注射4周后，ApoE-/-小鼠肝组织间隙绿色荧光显著减少，说明lunasin腹腔给药治疗4周后可显著抑制ApoE-/-小鼠肝组织分泌PCSK9蛋白。
上述实验结果证明，多肽lunasin腹腔给药后可显著抑制ApoE-/-小鼠肝脏组织表达和分泌PCSK9蛋白。
SEQUENCE LISTING
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Claims (9)
1.一种抑制前蛋白转化酶枯草溶菌素9 (PCSK9) 表达的方法，其特征在于使用露那辛抑制前蛋白转化酶枯草溶菌素9的产生，降低前蛋白转化酶枯草溶菌素9的血清水平，所述露那辛的氨基酸序列如SEQ ID No：1所示。
2.如权利要求1所述的方法，其特征在于所述露那辛氨基酸C末端是非酰胺化或酰胺化的。
3.如权利要求1所述的方法，其特征在于所述露那辛通过基因工程方法、化合合成方法或者从天然植物材料中提取的方法制备得到。
4.如权利要求1-3任一项所述的方法，其特征在于将露那辛与其它前蛋白转化酶枯草溶菌素9抑制剂联合使用。
5.如权利要求4所述的方法，其特征在于所述前蛋白转化酶枯草溶菌素9抑制剂包括PCSK9单克隆抗体、PCSK9小干扰RNA、PCSK9基因沉默寡核苷酸、小檗碱及其衍生物、姜黄素、海藻寡糖中的一种或几种。
6.一种露那辛的新用途，其特征在于露那辛作为前蛋白转化酶枯草溶菌素9抑制剂用于制备治疗前蛋白转化酶枯草溶菌素9介导的相关疾病的药物。
7.如权利要求6所述的用途，其特征在于所述前蛋白转化酶枯草溶菌素9介导的相关疾病包括高胆固醇血症、肥胖、糖尿病、肾病。
8.如权利要求6所述的用途，其特征在于所述露那辛可制备成药学上接受的任何一种制剂。
9.如权利要求8所述的用途，其特征在于所述露那辛可制备成片剂、胶囊、滴剂、粉针剂或水针剂。
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