CN114288302B - 一种增强剂3C1在制备FXR蛋白和Caspase 8蛋白相互作用的增强剂中的应用 - Google Patents
一种增强剂3C1在制备FXR蛋白和Caspase 8蛋白相互作用的增强剂中的应用 Download PDFInfo
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Abstract
本发明公开了一种增强剂3C1在制备FXR蛋白和Caspase 8蛋白相互作用的增强剂中的应用，该增强剂可促进FXR蛋白与Caspase 8蛋白之间的相互作用，进而抑制Caspase8向凋亡复合体的募集，阻断凋亡复合物形成，抑制Caspase 8的剪切活化，阻断凋亡进程，抑制细胞凋亡。因此，该增强剂可用于制备治疗具有凋亡表型的肝脏疾病的药物，具有良好的市场应用前景。
Description
技术领域
本发明涉及一种增强剂在制备蛋白相互作用增强剂中的应用，具体涉及一种增强剂3C1在制备FXR蛋白和Caspase 8蛋白相互作用的增强剂中的应用。
背景技术
目前，各种急慢性肝脏疾病(药物性肝损伤、酒精性脂肪肝、非酒精性脂肪肝、胆汁淤积型肝损伤、病毒性肝炎、肝纤维化、肝硬化和肝癌等)，在全球范围的发病率居高不下，是威胁人类健康的重要杀手之一。目前全球约有1.85亿人感染丙型病毒性肝炎；非酒精性脂肪性肝病(Nonalcoholic fatty liver disease，NAFLD)患病率超过25％，且已有超过1亿人口的进展为非酒精性脂肪性肝炎(Nonalcoholic steatohepatitis，NASH)。然而除了丙型病毒性肝炎外，目前可用于其他各类肝脏疾病治疗的特异性药物并不多。
细胞凋亡是一种基本的生物学现象，对生物体进化/发育和内环境稳态等具有重要作用；而细胞凋亡紊乱则与多种疾病的发生发展密切相关，如肝细胞凋亡是各种肝病(包括药物性肝损伤、酒精性肝损伤、NALFD/NASH、病毒性肝炎、胆汁淤积型肝损伤、缺血-再灌注性肝损伤、肝豆状核变性、肝纤维化/硬化、肝癌等)的共同特征和病理机制。抑制肝细胞凋亡被广泛认为是肝脏疾病治疗的重要策略之一(Cellular and molecular mechanismsof liver injury.Gastroenterology，2008，134(6)：1641-54.)。
法尼醇X受体(Farnesoid X Receptor，FXR，又称NR1H4)是核受体超家族的一员，在胆汁酸、糖、脂代谢中发挥重要功能，同时与能量平衡、炎症、细胞命运、纤维化和肝再生等生理病理过程密切相关。因此，FXR被广泛认为是重要的保肝药物研发靶标(FXRmodulators for enterohepatic and metabolic diseases.Expert OpinTherPat.2018Nov；28(11)：765-782.)。目前基于FXR为靶点的药物研发策略主要集中于FXR激动剂或拮抗剂的研发，以期调控FXR的转录活性。奥贝胆酸是一种FXR激动剂，于2016年5月被美国FDA批准上市用于抗原发性胆汁性肝硬化的治疗，是以FXR为靶点成功上市的第一例药物；其对NASH的作用已完成临床III期研究，然而其临床药效欠佳(Obeticholic acid forthe treatment of non-alcoholic steatohepatitis：interim analysis from amulticentre，randomised，placebo-controlled phase 3trial.Lancet.2019Dec 14；394(10215)：2184-2196.)。
研究表明，NASH以及肝纤维化状态下伴有显著的肝细胞凋亡。FXR激动剂对NASH等疾病状态下显著升高的肝细胞凋亡状态没有改善作用，是其临床药效局限的主要原因之一(Combined obeticholic acid and apoptosis inhibitor treatment alleviates liverfibrosis.Acta Pharm Sin B.2019May；9(3)：526-536.)。另外，项目组前期研究表明，肝细胞中FXR蛋白与Caspase 8蛋白之间具有稳定的蛋白-蛋白相互作用。当肝细胞受到凋亡刺激时，FXR与Caspase 8蛋白之间的相互作用减弱，使得Caspase 8与FADD、RIP1相互结合形成凋亡复合体，该复合体促进Caspase 8的剪切活化，促进凋亡的进程(Noncanonicalfarnesoid X receptor signaling inhibits apoptosis and impedes liverfibrosis.EBioMedicine.2018Nov；37：322-333.)。因此，稳定和增强FXR蛋白与Caspase 8蛋白之间的相互作用，是阻断凋亡信号传导及凋亡发生发展的重要策略。然而，FXR蛋白与Caspase 8蛋白之间的相互作用如何调控尚且未知，目前亦没有FXR-Caspase 8蛋白互作调控剂。
发明内容
发明目的：本发明的目的在于提供一种增强剂3C1在制备FXR蛋白和Caspase 8蛋白相互作用的增强剂中的应用。本发明还有一个目的是提供增强剂3C1作为FXR蛋白和Caspase 8蛋白相互作用的增强剂在制备治疗肝纤维化、肝损伤的药物中的应用。
技术方案：本发明所述的如式(1)所示的增强剂3C1在制备FXR蛋白和Caspase 8蛋白相互作用的增强剂中的应用：
所述增强剂3C1促进生理或病理状态下FXR蛋白和Caspase 8蛋白之间的相互作用。
所述增强剂通过阻断Caspase 8蛋白向凋亡复合体的募集，阻断凋亡复合体的形成。
所述增强剂阻断Caspase 8蛋白的剪切活化。
所述增强剂添加药学上可接受的辅料制备成制剂。
所述药学上可接受的辅料包括稀释剂、黏合剂、崩解剂、助流剂、润滑剂、矫味剂、包合材料、吸附材料。
所述制剂为颗粒剂、散剂、片剂、胶囊剂、丸剂、口服液。
所述增强剂3C1作为FXR蛋白和Caspase 8蛋白相互作用的增强剂在制备对抗肝细胞凋亡的药物中的应用。
所述增强剂3C1作为FXR蛋白和Caspase 8蛋白相互作用的增强剂在制备保护肝脏的药物中的应用。
所述增强剂3C1作为FXR蛋白和Caspase 8蛋白相互作用的增强剂在制备治疗肝纤维化和肝损伤的药物中的应用。
有益效果：与现有技术相比，本发明具有如下显著优点：(1)该增强剂能够增强生理状态下FXR蛋白与Caspase 8蛋白之间的相互作用，与对照组具有显著差异，最优达到***P＜0.001；(2)该增强剂能够恢复并增强凋亡状态下削弱的FXR蛋白与Caspase 8蛋白之间的相互作用；(3)该增强剂能够对抗各种诱因导致的肝细胞凋亡，与对照组具有显著差异，最优达到***P＜0.001；(4)该增强剂作用靶点精准、特异性高、专一性强，适应症广泛，可制备为治疗具有肝细胞凋亡表型的多种肝脏疾病的药物。
附图说明
图1为细胞凋亡状态下FXR蛋白与Caspase 8蛋白相互作用；
图2为本申请的增强剂3C1对健康细胞中FXR蛋白与Caspase 8蛋白相互作用的调控；
图3为本申请的增强剂3C1对凋亡细胞中FXR蛋白与Caspase 8蛋白相互作用的调控；
图4为本申请的增强剂3C1对细胞凋亡的保护作用，(A)ActD/TNFα引起的肝细胞凋亡；(B)CHX/FasL引起的肝细胞凋亡；(C)TRAIL引起的肝细胞凋亡；其中：*P＜0.05，**P＜0.01，***P＜0.001；
图5为本申请的增强剂3C1对CCl4引起的肝损伤的保护作用，(A)血清ALT水平；(B)血清AST水平；(C)肝脏H&E染色；(D)肝脏Sirius red染色；(E)肝脏Timp1的mRNA水平；(F)肝脏Timp2的mRNA水平；其中：*P＜0.05，**P＜0.01，***P＜0.001。
具体实施方式
实施例1
细胞凋亡状态下FXR蛋白与Caspase 8蛋白的相互作用变化
1、实验材料
DMEM培养基购自GIBCO(Grand Island，New York，USA)。胎牛血清(Fetal bovineserum，FBS)购自Hyclone(Logan，Utah，USA)。胰蛋白酶Trypsin购自Amersco(Solon，Ohio，USA)。细胞培养耗材购于Costar(USA)。放线菌素D(Actinomycin，ActD)和放线菌酮(cycloheximide，CHX)购于MedChem Express公司(NJ，USA)。重组人肿瘤坏死因子α(TumorNecrosis Factor alpha，TNFα)购于Peprotech公司(Rocky Hill，USA)，重组人FasL蛋白和重组人TRAIL蛋白购于Sino Biological公司(Wayne，PA，USA)。
2、实验方法
2.1细胞培养及给药处理
人肝细胞系L02培养于含10％胎牛血清的DMEM完全培养基，置于37℃，5％CO2的细胞培养箱中培养，取对数生长期的细胞按2×105个/孔接种于12孔板中。24h后分别用TRAIL，ActD/TNFα，CHX/FasL法造凋亡模型。TRAIL给药浓度为50ng/ml，给药时间为0、2、4、8、12、24h。ActD/TNFα给药浓度为0.2μM和20ng/ml，其中ActD预给30min，给药时间为0、1、2、4、8、12h。CHX/FasL给药浓度为50μM和50ng/ml，其中CHX预给30min，给药时间为0、2、4、8h。
2.2免疫共沉淀(Co-IP)
1)用预冷的PBS洗涤细胞两次，最后一次加入适量预冷的PBS，用预冷的细胞刮子将细胞从培养皿或培养瓶上刮下，把悬液转到1.5ml的EP管中。4℃，4000rpm离心5min后弃掉上清液，并加入适量NP40裂解液，振荡均匀并通过超声破碎细胞，超声5s/次，共3次，至细胞裂解完全。而后4℃，14000g离心10min，收集上清至新的EP管，即为蛋白裂解液。BCA法定蛋白浓度。
2)取25μl的ProteinA Beads用5％BSA室温封闭1h，而后用PBS洗3次，每次14000g离心1 min，弃上清。将浓度均一化的蛋白裂解液加入至Beads中，4℃摇转1h，将样品Pre-Clear。14000g离心1min，转移上清至新的EP管中。将Caspase 8抗体及IgG加至蛋白裂解液中，4℃摇床过夜，制备抗原-抗体结合物，次日洗Beads 2次后，于14000g，4℃离心1min，而后向Beads中加入Ag-Ab混合液，4℃摇床3h，制备Beads-抗原-抗体复合物。而后14000g，4℃离心1min。弃上清，留底(Beads)。洗Beads 3次，瞬时离心。向Beads中加入2×LoadingBuffer(30μl)，沸水煮5min，12000rpm，离心5min。进行Western blot和凝胶成像分析。
2.3 Western blot
1)细胞收集后置于冰上，每20μl细胞压积中加入100μl的RIPA细胞裂解液，1％Protease Inhibitor Cocktail(Sigma)，1％磷酸酶抑制剂，充分吹散团块，冰上裂解30min后，超声破碎5次，每次2s，期间暂停数秒以待裂解液冷却。13000g离心10min，所得上层清液为提取到的总蛋白样品，移入新离心管中，使用BCA法测定蛋白含量，加入4×loadingbuffer，混匀后煮沸10min待上样。
2)制备3％-5％浓缩胶和6％-12％分离胶的SDS-PAGE，电泳分离蛋白样本。将样本充分振匀，每样取50-100μg相同量的总蛋白进行上样。电泳以浓缩胶恒压75V 40min，分离胶恒压115V 60min梯度进行。使用半干转条件进行蛋白转印，将滤纸、切下凝胶和PVDF膜置于半干转缓冲液中平衡，滤纸平放于半干转槽中，上面依次放置PVDF膜，凝胶和另一份滤纸，并小心排净所有层与层间的气泡。将半干转的盖子扣在槽上，设置恒压25V，转印30min。
3)转印后PVDF膜在TBST溶液配制的5％脱脂奶粉封闭液中封闭，于37℃封闭1h。以100μl/cm2 PVDF的相对量加入封闭液配制的适量一抗，4℃孵育过夜。用TBST溶液漂洗PVDF膜4次，每次5min。将膜与HRP-结合的二抗稀释液于37℃孵育60min，用TBST溶液漂洗PVDF膜4次，每次5min。
4)使用ChemiDocTM XRS+凝胶成像系统(Bio-Rad)曝光，并用Image Lab TMsoftware进行光密度分析和半定量比较。
3、实验结果
如图1所示，生理情况下，FXR蛋白与Caspase 8蛋白之间具有相互作用，而在ActD/TNFα诱导的凋亡模型下，FXR与Caspase 8之间的相互作用减弱。
实施例2
增强剂3C1对FXR蛋白和Caspase 8蛋白之间相互作用的调控
1、实验材料
化合物3C1，购自陶素生物，结构式如下：
其余试剂同实施例1。
2、实验方法
2.1细胞培养及给药处理
人肝细胞系L02培养于含10％胎牛血清的DMEM完全培养基，置于37℃，5％CO2的细胞培养箱中培养
2.2化合物3C1对生理状态下FXR-Caspase 8蛋白互作的调控作用
取对数生长期的细胞按2×105个/孔接种于6孔板中。24h后以给予不同浓度的化合物3C1：1 μM、10μM、100μM，2h后收集细胞。
2.3化合物3C1对凋亡状态下FXR-Caspase 8蛋白互作的调控作用
取对数生长期的细胞按2×105个/孔接种于6孔板中。凋亡的诱导方法同实施例1。给予凋亡诱导剂的同时，给予不同浓度的化合物3C1：1μM、10μM、100μM，2h后收集细胞。
2.4Co-IP
同实施例1。
2.5 Western blot
同实施例1。
3、实验结果
3.1化合物3C1对生理状态下FXR-Caspase 8蛋白互作的调控作用
如图2所示，在正常细胞中，给予化合物3C1后，可以明显增强FXR蛋白与Caspase 8蛋白之间的相互作用，且此作用呈现剂量依赖性。
3.2化合物3C1对凋亡状态下FXR-Caspase 8蛋白互作的调控作用
同实施例1所述，凋亡状态下FXR-Caspase 8蛋白互作的下调。如图3所示，ActD/TNFα诱导的L02凋亡状态下，FXR蛋白与Caspase 8蛋白之间的相互作用减弱；而化合物3C1对此具有显著的逆转作用。
上述结果证实，化合物3C1为FXR和Caspase 8蛋白相互作用增强剂。
实施例3
增强剂3C1对细胞凋亡的药理活性
1、实验材料
噻唑蓝(Methylthiazolyldiphenyl-tetrazolium bromide，MTT)购于MedChemExpress公司(NJ，USA)。
其余试剂同实施例1。
2、实验方法
2.1细胞培养、转染和给药处理
人肝细胞系L02培养于含10％胎牛血清的DMEM完全培养基，置于37℃，5％CO2的细胞培养箱中培养，取对数生长期的细胞按5×103个/孔接种于96孔板中。24h后以50nM转染细胞，转染48h后进行凋亡造模。具体方法如下：TRAIL给药浓度为50ng/ml，给药24h。ActD/TNFα给药浓度为0.2μM和20ng/ml，其中ActD预给30min，给药时间为8h。CHX/FasL给药浓度为50μM和50ng/ml，其中CHX预给30min，给药时间为8h。
细胞给予凋亡试剂的同时，给予不同浓度的化合物3C1：1μM、10μM、100μM。
2.2细胞活力检测
给药时间结束后，每孔加入20μl MTT(5mg/ml)，37℃继续孵育4h，吸出弃去培养基，每孔加入150ld DMSO，37℃孵育10min，490nm波长测定吸光度。
3、实验结果
如图4所示，FXR-Caspase 8蛋白互作增强剂3C1在TRAIL(图4A)、ActD/TNFα(图4B)和CHX/FasL(图4C)3种经典凋亡模型下均具有显著的抗细胞凋亡效果，且呈现剂量依赖性。
实施例4
增强剂3C1对CCl4引起的肝损伤的保护作用
1、实验材料
实验小鼠(C57BL/6)购自北京维通利华实验动物技术有限公司；
CCl4购自上海凌峰化学试剂公司，矿物油购置Sigma-Aldrich公司，TrizolRNAiso plus购自Takara公司，逆转录试剂盒和SYBR Green购自Applied Biosystems公司，所用引物由Invitrogen公司(上海，中国)设计合成。DEPC水购于碧云天生物技术研究所(上海，中国)。
其余生物材料/试剂均可从市售途径获得。
其余实验材料同实施例1。
2、实验方法
2.1增强剂3C1对对CCl4诱导的小鼠肝损伤的作用
实验前C57BL/6小鼠适应性饲养一周，室温22～26℃，相对湿度40％～60％，明暗交替12h。所有小鼠分笼饲养，自由饮水采食，体重维持在20-22g。本实验分为空白对照组、模型组、3C1低剂量给药组(5mg/kg)和3C1高剂量给药组(10mg/kg)。
3C1低剂量给药组和3C1高剂量给药组中C57BL/6小鼠进行预给药处理，腹腔注射5mg/kg/day或10mg/kg/day 3C1，其余组别给予相同剂量的溶媒对照。3天后进行CCl4造模，空白对照组给予相同剂量的矿物质油。48h后安乐处死小鼠。收集小鼠血液和肝脏。
2.2血清转氨酶检测
全血室温静置0.5h后，8000rpm离心10min，转上清至新的EP管得到血清。按照ALT和AST试剂盒说明书对血清ALT和AST进行检测。
2.3 RT-PCR
2.3.1动物组织样本总RNA提取
1)分别称取各小鼠的肝组织样本20mg，加入1.0ml的RNAiso Plus，经均质器匀浆，将匀浆液转移至离心管中，室温静置5min，于4℃，12000g离心5min。
2)转上清800μl，并加入160μl氯仿，剧烈振荡15sec，室温放置5min后12000g离心15min。样品分为三层：底层为黄色有机相，上层为无色水相和一个中间层。
3)小心转移上层水相300μl到新管中，并加入300μl异丙醇，颠倒混匀后，室温放置10min，而后12000g离心10min，弃去上清；
4)用预冷的75％乙醇1.0ml洗涤RNA沉淀，而后12000g离心5min，弃上清得到总RNA，并用20μl DEPC水复溶，定量后稀释至0.5μg/μl。
2.3.2逆转录
按照说明书要求的体系配比将RNA溶液和试剂盒成分配制成总体积为20μl的体系并设定程序温度进行逆转录，具体配比要求如下：
2.3.3 PCR
PCR体系如下：
PCR使用条件如下：
Stage 1：预变性95℃1min
Stage 2：PCR反应95℃15sec；如60℃30sec for 40Cycles；72℃30sec
Stage 3：融解曲线分析65-95℃，0.5℃/5
2.3.4荧光定量PCR所使用的引物见下表1：
表1荧光定量PCR所使用的引物
2.4肝脏病理学分析
本部分H&E染色和Sirius Red染色均委托武汉谷歌生物技术公司完成。
3实验结果
3.1增强剂3C1对血清生化指标的作用
如图5A和5B所示，CCl4可引起小鼠血清ALT和AST水平显著升高，而增强剂3C1则可显著降低由CCl4注射引起的血清ALT和AST水平的升高。
3.2增强剂3C1对肝脏病理的改善作用
据肝脏病理学分析H&E染色结果(图5C)，对照组小鼠肝小叶结构完整，肝细胞围绕中央静脉放射状排列，无纤维增生及炎性细胞；肝细胞体积大，核圆，居中。而注射CCl4后肝血窦明显充血扩张，肝细胞发生颗粒样变，大量有部分肝细胞坏死灶，部分肝细胞水肿变性，炎性细胞浸润，部分肝内肝小叶结构破坏，汇管区纤维组织异常增生，胶原纤维增生明显，呈带状增生。3C1给药组肝小叶结构紊乱情况得到显著的改善，炎性细胞浸润减少，汇管区胶原纤维较少。表明3C1具有明显的肝保护作用。
据肝脏病理学分析Sirius Red染色结果(图5D)，对照组肝脏只有较少的红色胶原纤维染色，而注射CCl4后大量的红色胶原纤维存在于汇管区，3C1给药后，肝脏中红色胶原纤维增生情况得到显著的改善。
3.3增强剂3C1对肝脏纤维化基因的改善作用
据实时RT-PCR的结果显示(图5E和5F)，在CCl4诱导小鼠急性肝损伤模型中，肝脏纤维化基因Timp1和Timp2 mRNA水平显著升高(P＜0.001)，增强剂3C1可以显著降低Timp1和Timp2的mRNA的水平(P＜0.001)，且呈现剂量依赖性。
综合上述结果，提示增强剂3C1具有确切的肝脏保护作用。
实施例5
增强剂3C1 50mg，与微晶纤维素50mg、淀粉60mg混合，用适量20％乙醇作湿润剂，制成软材，常规方法制粒，加入滑石粉、硬脂酸镁混合，压片，成片剂。
增强剂3C1 50mg，与淀粉50mg、糊精10mg、蔗糖10mg混合，用30％乙醇作润湿剂，制成软材，湿法制粒，制成颗粒剂。
增强剂3C1 50mg，与淀粉50mg、糊精10mg、蔗糖10mg混合，用30％乙醇作润湿剂，制成软材，湿法制粒，然后装胶囊，制成胶囊剂。
增强剂3C1 50mg，与淀粉40mg、滑石粉10mg混合采用常规技术制成散剂。
增强剂3C1 50mg，与糖粉15mg、水8mL采用常规技术制成糖浆剂，再加入淀粉70mg采用常规技术制备成丸剂。
增强剂3C1 50mg，与聚维酮、蔗糖、水采用常规技术制成口服液。
Claims (4)
1.一种如式（1）所示的增强剂3C1作为FXR蛋白和Caspase 8蛋白相互作用的增强剂在制备治疗伴有肝细胞凋亡的肝纤维化和肝损伤的药物中的应用：
式（1）。
2.根据权利要求1所述的应用，其特征在于，所述增强剂添加药学上可接受的辅料制备成制剂。
3.根据权利要求2所述的应用，其特征在于，所述药学上可接受的辅料包括稀释剂、黏合剂、崩解剂、助流剂、润滑剂、矫味剂、包合材料、吸附材料。
4.根据权利要求2所述的应用，其特征在于，所述制剂为颗粒剂、散剂、片剂、胶囊剂、丸剂、口服液。
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