CN113101290A - mTOR抑制剂Torin1在制备胆汁淤积性胆管损伤药物中的应用 - Google Patents
mTOR抑制剂Torin1在制备胆汁淤积性胆管损伤药物中的应用 Download PDFInfo
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Abstract
本发明涉及生物技术领域,具体是mTOR抑制剂Torin1在制备胆汁淤积性胆管损伤药物中的应用,mTOR是一种进化保守的丝氨酸/苏氨酸激酶,在细胞生长和代谢中发挥关键作用。Torin 1是mTOR的抑制剂。mTOR通过形成两个多蛋白复合物mTORC1和mTORC2来调控细胞的生长、运动和代谢,mTORC1对营养物质敏感,mTORC2通过PI3K和生长因子信号通路调控。本发明发现Torin 1对胆汁淤积性胆管损伤具有显著治疗作用,治疗后可明显改善患有胆汁淤积性胆管损伤的肝功能指标以及炎性细胞浸润、胆管增生、胆管周肝纤维化等病理变化;此抑制剂能够明显降低胆汁淤积性胆管损伤动物模型中炎症因子水平。
Description
技术领域
本发明属于生物技术领域,具体涉及mTOR抑制剂Torin1在制备胆汁淤积性胆管损伤药物中的应用。
背景技术
原发性硬化性胆管炎(Primary sclerosing cholangitis,PSC)是一类胆汁淤积性胆管损伤因起的慢性进行性胆管疾病,其特征为肝内外胆管进行性炎症和纤维化,进而导致多灶性胆管狭窄等。大多数患者最终发展为肝硬化、门静脉高压和肝功能失代偿。目前尚无有效的治疗药物。胆管增生(Bile duct hyperplasia)与胆管活化是该病的早期表现,胆管上皮细胞分泌不同的细胞因子和相关蛋白,进而诱发胆管周围纤维组织增生导致胆汁淤积性肝纤维化,纤维组织增生又刺激胆管细胞形态发生了肥大、化生、萎缩、消失等改变,很多在汇管区的增生和纤维化病灶逐渐连接起来,形成肝硬化的。
胆汁淤积性胆管损伤动物模型的建立为胆汁淤积性胆管损伤的基础研究、治疗研究以及筛选防治胆汁淤积性胆管损伤的药物研究提供了一条途径。慢性DDC(3,5-Diethoxycarbonyl-1,4-dihydrocollidine,3,5-二乙氧基羰基-1,4-二氢-尿嘧啶)喂食是一种异生物诱导的胆汁淤积性胆管损伤模型,该模型主要是用含有0.1% DDC的饲料喂食小鼠,DDC喂食后可以导致卟啉类物质的分泌增加,形成卟啉栓塞,使胆管上皮细胞及肝细胞发生异常增生、变性坏死,并使血管粘附分子、骨桥蛋白(OPN)、肿瘤坏死因子α(TNF-α)等在胆管上皮细胞中的表达增加,最终形成胆管周炎性细胞增加和胆管增生,可以模拟PSC的发病历程,是目前公认的研究PSC发病机制的理想模型。该动物模型的建立为胆汁淤积性胆管损伤的基础研究、治疗研究以及筛选防治胆汁淤积性胆管损伤的药物研究提供了一条途径。
发明内容
本发明所要解决的技术问题Torin 1抑制mTOR信号通路,可以减轻在DDC诱导的胆管损伤中的作用,具体所述的胆管损伤类型为胆汁淤积性胆管损伤。通过Torin 1抑制mTOR信号通路,拟阐明Torin1在DDC诱导的胆管损伤中的治疗效果。
具体的,mTOR抑制剂Torin1在制备胆汁淤积性胆管损伤药物中的应用。
作为本发明所述的mTOR抑制剂Torin1在制备胆汁淤积性胆管损伤药物中的应用的优选方案:所述mTOR通过形成两个多蛋白复合物mTORC1和mTORC2来调控细胞的生长、运动和代谢。
作为本发明所述的mTOR抑制剂Torin1在制备胆汁淤积性胆管损伤药物中的应用的优选方案:所述Torin1是mTOR复合物mTORC1和mTORC2的抑制剂。
作为本发明所述的mTOR抑制剂Torin1在制备胆汁淤积性胆管损伤药物中的应用的优选方案:所述Torin1在2 nM和10 nM浓度下分别抑制mTORC1 和mTORC2底物的磷酸化。
一种治疗胆汁淤积性胆管损伤的药物,含有所述mTOR的抑制剂Torin1。
所述胆汁淤积性胆管损伤治疗药物中还含有药学上可接受的载体。
所述药学上可接受的载体,是指适用于人和/或动物而无过度不良副反应(如毒性、刺激和变态反应)即有合理的效益/风险比的物质。
此抑制剂Torin1在作为胆汁淤积性胆管损伤药物中进行制剂使用时,所述Torin1 是一种有效的 mTOR 抑制剂,IC50 为 3 nM。Torin 1 抑制 mTORC1/2 复合物。
与现有技术相比,具有如下有益效果:
经过广泛而深入的研究,首次发现了mTOR抑制剂Torin1对胆汁淤积性胆管损伤有显著治疗作用,以及首次发现mTOR抑制剂Torin1用于治疗胆汁淤积性胆管损伤中的新用途。
附图说明
图1、显示为Torin1改善DDC诱导胆汁淤积性胆管损伤。A:肝脏大体观;B:肝重/体重比;C:ALT;D:ALP;E:TBIL,与相应组别比,*p<0.05,***p<0.001。
图2、显示为Torin1改善 DDC诱导的胆汁淤积性胆管损伤肝脏病理变化(H&E染色)。黑色箭头指示胆管上皮细胞增生及炎性细胞浸润。
图3、显示为Torin1改善DDC诱导胆汁淤积性胆管损伤中胶原纤维沉积情况(Masson染色)A:各组小鼠肝脏胶原纤维沉积情况B:Masson染色图像分析结果,与相应组别比, *p<0.05,**p<0.01,***p<0.001。
图4、显示为Torin1降低DDC诱导胆汁淤积性胆管损伤中胆管上皮细胞的增生(CK19)。 A:DDC模型各组小鼠肝组织中CK19的分布 B:CK19表达的半定量分析,与相应组别比,**p<0.01,***p<0.001 。
图5、显示为Torin1降低DDC诱导胆汁淤积性胆管损伤中肝实质细胞的增生。A:DDC模型各组小鼠肝组织中Ki67的分布 B:Ki67表达的半定量分析,与相应组别比,*p<0.05,***p<0.001。
图6、显示为Torin1降低DDC诱导胆汁淤积性胆管损伤中促炎细胞因子的表达。qRT-PCR结果显示,与DDC组相比,DDC+Torin1组小鼠肝组织中Il6(A)、Mcp1(B)等促炎细胞因子的表达水平降低,Il10(C)、Arg1(D)等抑炎细胞因子的表达水平升高。与相应组别比,*p<0.05,**p<0.01,***p<0.001。
图7、显示为Torin1降低DDC诱导胆汁淤积性胆管损伤模型中相关蛋白的表达水平。A:Western blot检测mTOR、 P-mTOR、Akt、P-Akt(Ser473)、p65、P-p65 蛋白表达, B-D:相关蛋白的半定量分析 与相应组别比, *p<0.05,**p<0.01。
具体实施方式
实施例1:Torin1改善DDC诱导的胆汁淤积性胆管损伤
一、胆汁淤积性胆管损伤动物模型的建立和分组
实验动物:实验动物分组与模型的建立:6~8 周龄SPF级雌性 C57BL/6J小鼠平均体重20~30g,活动状态良好,饲养于无特定病原级的环境中。
实验小鼠分组
建立模型小鼠前,先要对纳入的实验小鼠进行分组,选取24只小鼠,按照随机分配法的原则,分为NMP(NMP,N-甲基吡咯烷酮,作为溶剂对照组)溶剂组、Torin1处理组、DDC+NMP组、DDC+Torin1组,每组小鼠6只。(说明:全文所述的Torin1均指mTOR抑制剂)。
诱导的胆管损伤动物模型建立
治疗组每周腹腔注射Torin1,两组均隔天注射,连续3 w。此期间对照组每天腹腔注射 0.1%(NMP)。Torin1按说明书溶于NMP,储存浓度为30 mg/ml,按照10 mg/kg体重的剂量每两天一次腹腔注射,最后一次注射 Torin1后24 h 进行取材及相关的检测。于3 w后剖杀小鼠,分离肝脏,肉眼观察小鼠肝脏大体病理变化,发现:NMP溶剂组和Torin1处理组:小鼠肝脏颜色鲜红,表面光滑,界面清晰,质软。DDC组小鼠肝脏淤血、肿大明显,颜色为黑褐色,表面有颗粒感,触之质韧。而(DDC+Torin1)组小鼠,肝脏淤血及肿大均明显减轻,颜色为暗红色,表面无颗粒感,质地稍韧(见图1A),其肝重/体重比较DDC组也得到明显改善(见图1B,p<0.05)。
二、样品收集与处理
收取肝组织样本
造模后3 w,眼球取血,采用颈法将其处死;用眼科剪从小鼠腹白线打开胸腔,将肝组织暴露开,用剪刀将肝脏的胆囊剔去,取下肝组织。分离血清,将眼球血用低温离心机3500 r/min、4℃离心15 min,分装,-80℃冰箱冻存备用。
将取出的肝组织置于冰上的玻璃块上,肝组织距离边缘4 mm处取肝组织一块(1cm×1 cm×1 cm),放入4%多聚甲醛溶液固定用于HE染色,将其余肝组织切割成若干块(1cm×1 cm×1 cm),于-80℃冰箱冻存备用。
血清中ALT、ALP、TBIL的检测
采集的血液静置凝固后,3 500 r/min离心15 min,分离血清,立即检测。血清中ALT、ALP、TBIL含量通过全自动生化分析仪测定,由徐州医科大学附属医院检验科提供。
结果如图1中显示,DDC给药后肝损伤明显,血清谷丙转氨酶(ALT)、碱性磷酸酶(ALP)和总胆红素(TBIL)含量均明显增高(p<0.001),而Torin1处理后,与DDC组相比,ALT含量(见图1C所示)、ALP含量(见图1D 所示,p<0.001)及TBIL含量均明显降低(见图1E所示,p<0.001)。结果表明Torin1可以明显降低DDC引起的肝胆损伤指标。
染色
为了研究DDC诱导C57BL/6小鼠导致的肝脏病理损伤,我们运用常规HE染色对肝脏进行病理学观察。结果如图2A所示, NMP溶剂组和Torin1处理组肝小叶完整,界限较为清晰,肝细胞结构完整饱满,未发现胆管增生;而DDC模型组中央静脉周围较多中性粒细胞、单核细胞等炎性细胞浸润,胆管增粗、变大,增生明显,胆管中有原卟啉栓塞形成,表明胆管发生梗阻并伴有增生。胶原纤维增多,可见较明显的纤维间隔,成纤维细胞在胆管周围聚集,表明有肝纤维化发生。而与DDC组相比,DDC+Torin1组肝脏中炎性细胞浸润减少,胆管增生减轻,胆管中卟啉栓塞数目减少,胆管周聚集的成纤维细胞数目减少(见图2A、B,p<0.05)。结果表明,Torin1处理后可明显降低DDC引起的炎性细胞浸润及胆管上皮细胞的增生。
染色
为了观察各组小鼠肝脏胶原纤维沉积情况,我们对各组小鼠肝脏组织进行Masson染色(Masson染色试剂盒购自南京建成生物工程研究所),并用普通光学显微镜观察小鼠肝脏组织Masson染色情况,结果如图3A所示:正常组小鼠(NMP溶剂组和Torin1处理组)肝小叶结构清晰完整,肝细胞以中央静脉为中心呈放射状排列,无胶原纤维增生,中央静脉及肝窦内汇管区血管壁少量染成蓝色的胶原纤维分布,见汇管区及中央静脉区血管壁着蓝色。模型组(DDC组)小鼠肝小叶结构紊乱,胶原纤维明显沉积,并且在扩张的胆管周围,沿着胆管的走势,有明显的蓝色沉积,经过Image J软件分析发现与对照组相比,蓝色面积明显增多,提示胶原纤维沉积增多发生在胆管周围。Torin1处理后则有明显好转,胆管周围胶原纤维的沉积面积减少,纤维化程度减轻(见图3B,p<0.05)。
免疫组化检测胆管上皮细胞增生情况
微波处理(0.01 mmol/L 柠檬酸盐缓冲液,pH6.0)石蜡切片(4μm厚),用5% BSA室温封闭样本组织30 min,使用单克隆小鼠抗CK19,Ki67抗体(稀释 1:100;Abcam), 对照组滴加1% BSA,置于湿盒中4℃过夜。于第二天取出玻片,滴加预先按说明书稀释的二抗,将玻片置于湿盒中,放于 37℃温箱静置30min。配制DAB显色液,滴加于切片组织上,镜下观察显色变化。于玻片上滴加苏木素染色剂覆盖组织,室温静置 5~10s,流水冲洗干净,脱水封片。于光学显微镜下观察阳性分布情况,并用ImageJ定量分析。
结果如图4、图5显示,与正常组相比,DDC饮食组CK19、Ki67表达明显增加,而Torin1可以降低DDC饮食小鼠肝脏CK19(图4A、B、p<0.05)、Ki67的表达(见图5A、B,p<0.05)。因此,本实例再次证实Torin1通过抑制mTOR信号缓解DDC诱导的胆管损伤。
小鼠肝组织中炎症因子的表达情况
检测促炎因子 (IL-6和 MCP1)和抑炎因子(IL-l0和Arg1)的mRNA 相对表达水平。用 Trizol 法按说明书步骤提取各组肝组织的总RNA,将RNA逆转录成cDNA后进行PCR扩增反应。以β-actin作为内参,目的基因mRNA表达量用2-∆∆Ct计算,引物序列见表1 。
qRT-PCR结果如图6显示,与DDC组相比,DDC+Torin1组小鼠肝组织中Il6(p<0.001)、Mcp1(p<0.01)等促炎细胞因子的表达水平降低,Il10(p<0.001)、Arg1(p<0.001)等抑炎细胞因子的表达水平升高。因此,Torin1可降低 DDC诱导小鼠肝脏炎症。
表1 本实施例中所用引物序列
Western blot 检测mTOR、P-mTOR、 Akt、P-Akt(Ser473)、p65、P-p65 蛋白及其磷酸化水平
提取各组细胞总蛋白,用BCA法测浓度,每孔取40 μg蛋白样品上样进行SDS-PAGE电泳,用Bio-Rad 标准转膜装置进行湿式转膜,5%脱脂牛奶室温封闭 2 h,一抗 4℃冰箱摇床孵育过夜,洗膜,二抗孵育室温2 h,洗膜,加 ECL 显影液曝光显影,用ImageLab 软件分析结果,将目的蛋白灰度值/对应内参蛋白灰度值的比值作统计学分析,实验重复3次。
结果显示DDC组P-mTOR、P-AKT和P-p65磷酸化水平明显升高,而DDC+Torin1组其磷酸化水平相比DDC组均明显降低(见图7,p<0.05)。因此,Torin1有效抑制了DDC诱导 Akt/mTOR/NF-κB通路的活化。
以上已对本发明创造的较佳实施例进行了具体说明,但本发明创造并不限于所述实施例,熟悉本领域的技术人员在不违背本发明创造精神的前提下还可做出种种的等同变型或替换,这些等同的变型或替换均包含本申请权利要求所限定的范围内。
Claims (4)
1.mTOR抑制剂Torin1在制备胆汁淤积性胆管损伤药物中的应用。
2.根据权利要求1所述的mTOR抑制剂Torin1在制备胆汁淤积性胆管损伤药物中的应用,其特征在于:所述mTOR通过形成两个多蛋白复合物mTORC1和mTORC2来调控细胞的生长、运动和代谢。
3.根据权利要求2所述的mTOR抑制剂Torin1在制备胆汁淤积性胆管损伤药物中的应用,其特征在于:所述Torin1是mTOR复合物mTORC1和mTORC2的抑制剂。
4.根据权利要求2所述的mTOR抑制剂Torin1在制备胆汁淤积性胆管损伤药物中的应用,其特征在于:所述Torin1在2 nM和10 nM浓度下分别抑制mTORC1 和mTORC2底物的磷酸化。
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