CN114224886A - 奎纳克林在制备治疗肝纤维化药物中的应用 - Google Patents
奎纳克林在制备治疗肝纤维化药物中的应用 Download PDFInfo
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- CN114224886A CN114224886A CN202111308135.0A CN202111308135A CN114224886A CN 114224886 A CN114224886 A CN 114224886A CN 202111308135 A CN202111308135 A CN 202111308135A CN 114224886 A CN114224886 A CN 114224886A
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Abstract
Description
技术领域
本发明涉及奎纳克林在制备治疗肝纤维化药物中的应用,属于化学药物开发技术领域。
背景技术
肝纤维化是全球范围内死亡的主要原因之一,与异常比例的细胞外基质(ECM)的过度积累有关。肝纤维化导致肝结构紊乱,可能演变为肝硬化或肝癌。在肝纤维化的早期,受损的肝细胞和内皮细胞释放趋化因子以募集、产生巨噬细胞和各种炎症性细胞因子,其中包括转化生长因子β(TGF-β)。
TGF-β 超家族中分为四种亚型(TGF-β1、TGF-β2、TGF-β3、TGF-β1 β2 ),该家族成员参与调控细胞分化、细胞衰老、细胞凋亡、细胞粘连与迁移、胞外基质合成与重塑、表皮细胞-间充质细胞转变(epithelial-mesenchymal transition,EMT)、免疫抑制剂和血管形成等病理生理过程,在早期胚胎发育、组织器官形成和成体稳态平衡中发挥着至关重要的作用。
TGF-β1 是一种具有多功能性的细胞因子,在细胞的增殖、迁移、凋亡以及组织的损伤修复、免疫调节等方面起到了重要作用。TGF-β1 /Smads信号通路由TGF-β1、TGF-β受体蛋白(TGFR)、Smads 蛋白家族及相关调控基因等组成,是纤维化类疾病中主要的调控机制。TGF-β在未激活前以前体LTGF-β形式存在于细胞内,在组织纤维化过程中,TGF-β1 被激活并通过多种机制成为诱导纤维化的最强细胞因子。激活的TGF-β1与TGF-β受体2 (TGFR2)结合后招募并激活TGFR1。活化的TGFR1使Smad2和Smad3磷酸化并与Smad4形成复合物并转运到细胞核中,在细胞核内,该复合物的Smad3组分直接与基因启动子结合,诱导促纤维化分子的转录,包括α-平滑肌肌动蛋白(α-SMA)、I型胶原(COL1A)和基质金属蛋白酶组织抑制剂(TIMP)的表达,从而使肌成纤维细胞活化和ECM沉积。
肝纤维化主要是因各种致病因素(如病毒、异常代谢、大量饮酒和过度的免疫反应等)造成的,以细胞外基质过量积聚、肝星状细胞活化为特点的一种创伤愈合过程中的慢性肝损害性疾病。研究发现,肝纤维化受多种细胞内信号转导途径的控制,尤其以TGF-β1/Smads 信号通路最为重要。在肝脏内,TGF-β1/Smads信号通路参与了ECM的合成与降解、炎症反应和组织修复过程。肝纤维化发生后,TGF-β1 的分泌和表达与肝纤维化严重程度相关。研究显示,TGF-β1 可以抑制HSCs 的凋亡,诱导其生成大量基质蛋白(如纤维连接蛋白,I、III、IV型胶原蛋白)。一般情况下TGF-β1 在肝组织内表达量较少,当肝损伤后,HSCs 活化异常时TGF-β1 表达可显著增加。此外,TGF-β1 还能干扰降解ECM 的基质金属蛋白酶的产生;促进肝细胞凋亡;调控ECM 合成和降解、增加胶原沉积。因此,细胞外基质(ECM)产生的关键途径是通过TGF-β1 / Smads信号传导将静止的肝星状细胞(HSC)活化为肌成纤维细胞样细胞。所以TGF-β1/Smads信号传导通路可以作为抗肝纤维化的重要靶标。
奎纳克林是1920年代发现的,历史上曾被用作预防和治疗的抗疟药。奎纳克林是一种吖啶衍生物,可作为奎纳克林二盐酸盐口服给药 。奎纳克林二盐酸盐结构式如下:
奎纳克林还可用作贾第鞭毛虫病的抗菌药,系统性红斑狼疮和类风湿性关节炎的抗炎药,以及作为胸膜内硬化剂用于预防恶性胸腔积液和气胸的复发。此外,它在某些国家仍被用于女性绝育,并且正在针对克雅氏病进行临床评估。因其副作用小,毒性较低且大多数副作用在停药后可逆转,被用作抗癌潜在药物,已有相关研究发现其在结肠癌、非小细胞肺癌、前列腺癌、肾癌、头颈癌、白血病、乳腺癌、卵巢癌等中的抗癌潜力。
发明内容
本发明针对现有技术的不足,提供奎纳克林在制备治疗肝纤维化药物中的应用。
奎纳克林在制备治疗肝纤维化疾病药物中的应用,所述奎纳克林的结构式如下:
根据本发明优选的,所述肝纤维化疾病为由TGF-β1/Smads 信号转导通路异常导致的。
根据本发明优选的,所述奎纳克林作为药效成分。
根据本发明优选的,所述药物还包括促进剂,促进剂可以提高CUL4B蛋白与奎纳克林的结合。
一种适于肝部施用的药物,所述药物含有奎纳克林、奎纳克林对映异构体或非对映异构体、奎纳克林药学上可接受的盐做为药效成分。
根据本发明优选的,所述药物还含有提高CUL4B蛋白与奎纳克林结合的促进剂。
一种预防肝纤维化的保健品,所述保健品含有奎纳克林、奎纳克林对映异构体或非对映异构体、奎纳克林食品上可接受的盐做为有效成分。
根据本发明优选的,所述的保健品还含有提高CUL4B蛋白与奎纳克林结合的促进剂。
CUL4B蛋白可做为靶蛋白在筛选治疗肝纤维化疾病的药物中进行应用,所述药物与靶蛋白CUL4B结合后,能够增加靶蛋白CUL4B的稳定性。
原理说明
目前已有研究证实奎纳克林与化疗药物具有协同作用,可以增加肝癌细胞对化疗药物的敏感性,从而抑制肿瘤的增殖与生长。也有研究证实奎纳克林可以与其他抗癌药物联用从而提升药物对例如乳腺癌、卵巢癌、结直肠癌等的治疗效果,但发明人通过研究发现,肝纤维化受到TGF-β1/smads信号的调控,而虽然现有技术中有报道肝纤维化可发展为肝硬化,而肝硬化是原发性肝癌的重要辅助因子,但本领域技术人员始终认为奎纳克林在治疗肝癌的过程中仅仅是一种增敏剂,其并不对治疗肿瘤具有相关作用,也不会对TGF-β1/smads信号通路进行相关调节。
有益效果
本发明首次发现奎纳克林能够通过与CUL4B结合并保护其不被降解,进而影响TGF-β1/Smads 信号转导通路,实现治疗肝纤维化的效果,该发现克服了现有技术认为奎纳克林只是作为一种增敏剂参与到肝癌的治疗过程中,从而显著拓宽了奎纳克林的应用范围。
附图说明
图1为实施例1证明奎纳克林可以与CUL4B蛋白结合的DARTS实验的结果照片;
图2为实施例2细胞热转移实验(CETSA实验)的结果照片;
图3为实施例3不同剂量奎纳克林组及对照组小鼠体重变化曲线图;
图4为实施例3奎那克林组和对照组的小鼠血清中ALT和AST值的柱状图;
图5为实施例3奎纳克林组和对照组的小鼠肝脏组织中可溶性胶原含量的柱状图;
图6为实施例3中小鼠肝组织提取RNA使用RT-PCR检测结果的柱状图;
图7为实施例3中小鼠肝组织的HE染色、天狼星红染色及免疫组化染色结果的柱状图;
图8为实施例3中小鼠肝脏组织进行免疫组化实验观察P-SMAD2/3的表达情况的照片;
图9为实施例4中人肝星状细胞系LX-2的RT-PCR检测结果的柱状图;
图10为实施例5中提取细胞总蛋白后进行western blot实验检测结果的照片;
图11为实施例6中敲除CUL4B基因的稳转细胞系进行western blot实验检测结果的照片;
图12为实施例6中敲除CUL4B基因的稳转细胞系进行TGF-β刺激后 RT-PCR检测结果的柱状图。
具体实施方式
下面结合实施例对本发明的技术方案做进一步阐述,但本发明所保护范围不限于此。
实施例1
药物(奎纳克林)与靶蛋白(CUL4B)结合稳定性实验(DARTS实验)
1)配制DARTS细胞裂解液(1mL):M-PER哺乳动物蛋白质抽提试剂 730μL、蛋白酶抑制剂 10μL、200 mM 磷酸酯酶抑制剂sodium orthovanadate 10μL、1 M氟化钠溶液 50μL、100mM β-甘油磷酸钠溶液 100μL、50mM焦磷酸钠溶液 100μL;配制10×TNC(1 mL):1 MTris-HCl缓冲液,pH 8.0 500μL、5 M 氯化钠溶液 100μL、1 M 氯化钙溶液 100μL、无菌去离子水 300μL。使用时需用无菌去离子水将10×TNC稀释10倍;使用时链酶蛋白酶 Pronase的浓度为10mg/mL。
2)用预冷的磷酸盐缓冲盐溶液(PBS)洗涤LX-2细胞2次,吸净PBS溶液后加入500μLM-PER裂解液,刮取细胞并移至1.5mL EP管内,于4℃摇床孵育1.5小时后13000rpm、4℃离心15分钟,后吸取上清并分别于两组EP管内各加入225μL上清蛋白,一组加入5μL DMSO(药物溶剂,对照组),另一组加入5μL盐酸奎纳克林(药物终浓度为200μM,药物组),避光室温摇床孵育1.5h后将每组蛋白分为4管,(每管50μL),共8管药物溶液。使用1×TN稀释链酶蛋白酶Pronase,按照链酶蛋白酶 Pronase/上清蛋白质量为0、1:1600、1:800、1:400配制四种浓度的蛋白酶溶液两组,共8管蛋白酶溶液。然后将配制好的8管蛋白酶溶液分别加入对应的8管药物溶液的管内(-0、+0、-1:1600、+1:1600、-1:800、+1:800、-1:400、+1:400,-为对照组、+为药物组),室温孵育10min,每管加入1μL cocktail,冰上孵育5min。按照蛋白:loadingbuffer为4:1比例加入loading buffer,水浴锅煮沸10min。按上述顺序加样,十二烷基硫酸钠-聚丙烯酰胺凝胶(SDS-PAGE)电泳分离后行考马斯蓝染色1h,更换脱色液洗脱至出现清晰条带。
3)结果如图1所示,在100kDa左右出现一条差异条带,切出差异条带进行质谱分析,找出对应的差异蛋白-CUL4B,进行Western Blot验证,验证结果表明奎纳克林可以与CUL4B结合并可能通过其发挥抑制TGF-β1/Smads 信号转导通路的作用。
上述操作为本领域常规技术操作,如可参考《药物亲和反应的靶点稳定性(DARTS)技术研究进展—一种定位药物靶标的方法》(李德军等,《中兽医医药杂质》,2021年40卷第2期)等。
实施例2
细胞热转移实验(CETSA实验)
1)固定剂量不同温度下药物与靶蛋白的结合
取对数生长期的人肝星状细胞LX-2,使用培养基(H-DMEM+10%FBS+1%双抗)培养细胞密度至80%时,分别加入DMSO(对照组)与盐酸奎纳克林(药物终浓度为20μM,溶剂为DMSO,药物组),于培养箱中孵育2h。用预冷的PBS溶液洗涤细胞2次,吸净PBS溶液后再加入1mLPBS+10μLcocktail,用细胞刮刮取细胞并将细胞悬液均匀分配到200μL EP管中,共9管 ,每管100μL,按照温度为50℃、55℃、60℃、65℃、70℃、75℃、80℃、85℃、90℃分别标记EP管。使用96孔板热循环仪按照九个温度分别每管加热3min,到时后立即取出管子,室温孵育3min;然后置于液氮中快速冷冻。使用液氮和设置为25℃的热循环器将细胞冷冻-解冻两次,解冻后混匀管内细胞再进行下一循环,确保管子之间温度均匀。循环结束后4℃、2000g离心20min,使细胞碎片和聚集的蛋白质一起沉淀,取上清。取新EP管分别加入60μL上清液和15μLloading buffer,混匀后水浴锅煮沸10min。进行 Western Blot实验检测。
2)固定温度不同剂量下药物与靶蛋白的结合
同样取对数生长期的人肝星状细胞LX-2,使用培养基(H-DMEM+10%FBS+1%双抗)培养细胞密度至80%时,分别加入10μL不同浓度的盐酸奎纳克林,使盐酸奎纳克林终浓度分别为0、0.0001μM、0.001μM、0.01μM、0.1μM、1μM、10μM。然后按照1)中所述实验步骤再次进行实验操作,不同之处为此次7管不同浓度的细胞悬液均在同一70℃温度下于96孔板热循环仪进行3min加热。
3)通过细胞热转移实验量化目标蛋白在不同温度及不同奎纳克林浓度的条件下CUL4B蛋白热变性的变化,可以看到在不同温度(50-90℃)中,相比较于DMSO对照组,药物组均减少了CUL4B蛋白的变性,尤其以70℃时差异最为明显,表明盐酸奎纳克林可以与CUL4B蛋白结合并增加其热稳定性。同时,可以看到在70°C时,盐酸奎纳克林以剂量依赖的方式阻止CUL4B蛋白的变性(从属促进结合的促进剂),其EC50为0.07203。CTESA实验表明盐酸奎纳克林对CUL4B变性起抑制作用且呈药物剂量依赖性。
通过细胞热转移实验量化目标蛋白在不同温度及不同奎纳克林浓度的条件下CUL4B蛋白热变性的变化,可以看到在不同温度时,相比较于二甲基亚砜(DMSO,奎纳克林药物溶剂),奎纳克林可以减少CUL4B蛋白的变性,尤其是在70℃最为明显,并且在70℃时,奎纳克林以剂量依赖的方式阻止CUL4B变性(从属促进结合的促进剂),其EC50为0.07203,CTESA实验表明奎纳克林对CUL4B变性起抑制作用且呈药物剂量依赖性,结果如图2所示。
上述操作为本领域常规技术操作,如可参考《Monitoring Global Changes ofProtein Interaction States with the Proteome-Wide Cellular Thermal ShiftAssay (CETSA)》(Lingyun Dai,etc,《Annual Review of Biochemistry》,2019. 88:27.1–27.26)等。
实施例3
肝纤维化体内实验
实验材料:C57BL/6J小鼠,购自济南朋悦动物繁育有限公司;硫代乙酰胺,购自上海阿拉丁生化科技股份有限公司;盐酸奎纳克林,购自MedChemExpress。
实验方法:6-8w(周)的C57BL/6J小鼠, 硫代乙酰胺(TAA),每周三次腹腔注射,第1、2次50mg/kg,第3-5次100mg/kg,第6-10次200mg/kg,第11-15次300mg/kg,第16次以后400mg/kg,实验组第一天开始给小鼠灌喂盐酸奎纳克林(高浓度:25mg/kg和低浓度:10mg/kg),对照组第一天开始灌喂纯净水,8w后麻醉下脊椎脱臼处死小鼠,取血清和肝组织。血清检测ALT和AST。肝组织提取RNA,使用RT-PCR检测col1a1、col1a2、FN和ACTA2的表达量。肝组织制备石蜡切片,使用HE染色法、天狼星红染色法观察肝脏结构及肝脏纤维瘢痕形成程度,使用免疫组化法检测col1a和αSMA的表达量。
实验结果:小鼠体重变化如图3,对照组体重明显下降,奎那克林组体重下降缓慢;
小鼠血清ALT和AST变化如图4,奎那克林组ALT和AST相对于对照组明显下降(ALT正常值25-74IU/L,AST正常值23-48IU/L);提取肝组织用Biocolor可溶胶原蛋白检测试剂盒(Sircol Soluble Collagen Assay)检测可溶性胶原的含量,结果如图5所示,发现奎纳克林治疗组显著减少了TAA所引起的肝脏组织中胶原含量的升高。小鼠肝组织提取RNA使用RT-PCR检测结果如图6,结果表现为对照组纤维化相关指标升高,奎纳克林治疗后相关指标表达下降;小鼠肝组织制备石蜡切片后使用HE染色、天狼星红染色及免疫组化染色结果如图7,HE染色可见对照组肝组织肝小叶及汇管区结构不明显,形成假小叶样结构改变,奎纳克林治疗组较对照组肝组织结构改善明显。天狼星红三色染色显示对照组内大量胶原纤维沉积,呈红色,自汇管区周围向外延伸,较奎纳克林治疗组纤维条索明显增粗且着色较深,表明奎纳克林治疗组明显减少了胶原纤维的形成。免疫组化染色结果示奎纳克林治疗组肝组织的αSMA和col1a的表达量明显降低。以上结果充分证明在动物体内奎纳克林可以抑制肝脏纤维化后HSC的激活以及胶原纤维的形成。
通过对小鼠肝脏组织进行免疫组化实验观察P-SMAD2/3的表达情况,发现奎纳克林可以明显抑制肝脏纤维化模型小鼠中的SMAD2/3磷酸化,充分证明奎纳克林可以通过抑制TGF-β1/Smads信号通路从而影响肝脏纤维化的发生发展,如图8所示。
上述操作为本领域常规技术操作,如可参考《Milk Fat Globule-EGF Factor 8,Secreted by Mesenchymal Stem Cells, Protects Against Liver Fibrosis in Mice》(Su Yeon An, etc.《Gastroenterology》,017;152:1174–1186)等。
实施例4
人肝星状细胞系LX-2体外实验
实验材料:人肝星状细胞系LX-2,购自上海中乔新舟生物科技有限公司;TGF-β,购自PeproTech;盐酸奎纳克林,购自MedChemExpress。
实验方法:取对数生长期的人肝星状细胞系LX-2,接种到6孔板,置37℃、5%CO2及饱和湿度的培养箱中培养24h。待细胞长至70%融合度时,按分组情况,进行不同处理,奎那克林终浓度为1uM,TGFβ浓度为10ng/ml,置培养箱中继续培养,24小时后提取RNA,使用RT-PCR检测纤维化相关指标。
实验结果:LX-2细胞RT-PCR结果如图9所示,加入TGFβ刺激后纤维化指标上升,但在TGF刺激后加入奎那克林,指标则显著下降,且呈剂量依赖性,证明奎那克林可以治疗肝纤维化。
上述操作为本领域常规技术操作,如可参考《ECM1 Prevents Activation Q3 ofTransforming Growth Factor b,Hepatic Stellate Cells, and Fibrogenesis inMice》(Weiguo Fan, etc.《Gastroenterology》,2019;-:1–16)等。
实施例5
实验方法:取对数生长期的人肝星状细胞系LX-2,接种到6孔板中,置37℃、5%CO2及饱和湿度的培养箱中,培养细胞长至70%融合度时,对照组给予DMSO,药物组给予盐酸奎纳克林(药物溶剂为DMSO,终浓度10ng/mL),每孔加入TGF-β(10 ng/mL)刺激细胞,按照加入TGF-β后的0min、15min、30min、60min提取细胞蛋白,使用细胞裂解液提取总蛋白,通过Western Blot检测TGF-β通路的相关指标。
实验结果:如图10所示,奎纳克林明显抑制了总蛋白中P-Smad2/3的表达,而P-Smad2/3的蛋白表达是TGF-β1/Smads信号通路激活的表现。
实施例6
CUL4B敲除验证实验
CRISPR-Cas9系统:CRISPR/Cas9是一种基因精确编辑技术,使用该技术能够进行细胞水平单基因或多基因敲除,其原理是核酸内切酶Cas9蛋白通过导向性RNA(guide RNA,gRNA)对目标DNA序列的PAM依赖性识别并在PAM区(5’-NGG)上游3bp的特定位点启动DNA切割。Cas9核酸内切酶生成的双链断裂可通过同源介导的修复(HDR,Homology directedrepair)或非同源末端连接途径(NHEJ,Non-homologous end joining)进行修复。NHEJ修复会在DSB位点随机引入碱基的插入或者缺失(Indel),如果这些Indel不是3的倍数就会导致后续的阅读框发生移码,这种移码往往会产生提前终止密码子(premature terminationcodon,PTC),导致蛋白功能的丧失(提前翻译终止的多肽一般会被降解),实现基因敲除(KO,Loss of Function)。
使用CRISPR-Cas9系统敲除LX-2细胞CUL4B编码基因,使用Western Blot验证CUL4B编码基因的敲除情况,结果如图11所示,CUL4B编码基因敲除后LX-2细胞不再表达CUL4B,说明通过CRISPR-Cas9系统,成功在LX-2细胞系中实现CUL4B的敲除。
将CUL4B敲除细胞系(KO)、空转细胞系(KONC)两种稳转细胞系接种到6孔板中,37℃、5%CO2及饱和湿度的培养箱中培养细胞长至70%-80%时,KONC组和KO组给予DMSO,KONC+奎纳克林和KO+奎纳克林组给予TGF-β1(药物溶剂为DMSO,药物终浓度10μM),置于培养箱中继续培养6h。Western Blot和RT-PCR验证敲除靶蛋白CUL4B编码基因后是否还能抑制Smad2/3的激活以及纤维化的发展。
Western Blot结果如图11所示,敲除CUL4B后,加入盐酸奎纳克林已不能抑制LX-2细胞中Smad2/3的磷酸化,说明敲除了CUL4B蛋白,盐酸奎纳克林对TGF-β1/Smads信号转导通路的抑制作用也随之消失。
RT-PCR结果如图12所示,敲除CUL4B后,加入盐酸奎纳克林也无法抑制TGF-β1诱导的纤维化相关mRNA(ACTA2、FN、COL1A1)的高表达,说明靶蛋白CUL4B被敲除后,盐酸奎纳克林抑制纤维化的作用被取消。
以上结果均证明盐酸奎纳克林是通过与CUL4B蛋白结合然后抑制TGF-β1/Smads信号通路的激活从而具有治疗肝脏纤维化的作用。
Claims (10)
2.如权利要求1所述的应用,其特征在于,所述肝纤维化疾病为由TGF-β1/Smads 信号转导通路异常导致的。
3.如权利要求1所述的应用,其特征在于,所述奎纳克林作为药效成分。
4.如权利要求1所述的应用,其特征在于,所述药物还包括促进剂,促进剂可以提高CUL4B蛋白与奎纳克林的结合。
5.一种适于肝部施用的药物,所述药物含有奎纳克林、奎纳克林对映异构体或非对映异构体、奎纳克林药学上可接受的盐做为药效成分。
6.如权利要求5所述的药物,其特征在于,还含有提高CUL4B蛋白与奎纳克林结合的促进剂。
7.如权利要求5所述的药物,其特征在于,所述药物还含有药学上可接受的载体或者辅料。
8.如权利要求7所述的药物,其特征在于,所述药物的剂型包括片剂、粉剂、注射剂、胶囊剂或气雾剂。
9.一种预防肝纤维化的保健品,所述保健品含有奎纳克林、奎纳克林对映异构体或非对映异构体、奎纳克林食品上可接受的盐做为有效成分。
10.如权利要求9所述的应用,其特征在于,所述的保健品还含有提高CUL4B蛋白与奎纳克林结合的促进剂。
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