CN116942668A - Glyt1抑制剂在治疗器官纤维化中的应用 - Google Patents
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Abstract
本发明涉及GLYT1抑制剂在治疗器官纤维化中的应用,尤其在制备治疗器官纤维化药物中的应用;以GLYT1为靶点,通过GLYT1抑制剂抑制甘氨酸转运子GLYT1实现对纤维化器官的治疗,包含GLYT1抑制剂的药物尤其适合用于治疗肺纤维化。通过GLYT1抑制剂抑制甘氨酸转运子GLYT1,从而减少胶原合成,可以明显改善博来霉素引起的肺纤维化,对已经形成的肺纤维化也有明显的改善;并且以GLYT1为靶点,该方案对其它生理功能的影响较小。
Description
技术领域
本发明属于医药领域,尤其是涉及一种GLYT1抑制剂在治疗器官纤维化中的应用。
背景技术
脏器纤维化是器官组织内纤维结缔组织增多和实质细胞减少的病理变化,是多种慢性疾病的共同病理特征。纤维化可以影响任何器官,据统计,高达45%的死亡是由纤维化造成的。此外,与纤维化相关的常见疾病包括肝硬化、肝炎、非酒精性脂肪性肝炎、慢性肾脏疾病、心肌梗塞、心力衰竭、糖尿病、特发性肺纤维化和硬皮病等。出现纤维化相关疾病的患者约占重要脏器疾病发病人数的1/20。
因此,发现与人类纤维化疾病高度相关的关键治疗靶点,以及针对这些靶点开发高效的抗纤维化疗法是未来研究的重点。然而,尽管目前对纤维化病理学的理解取得了实质性进展,但尚缺乏有效治疗手段。
治疗器官纤维化的主要手段之一是应用大剂量的糖皮质激素以减少炎症反应,但存在股骨头坏死等较严重的副作用,对于非急性感染引起的慢性器官纤维化效果不佳。阻断TGF-β通路是治疗纤维化疾病的重要靶点,但是TGF-β信号传导在人体内分布广泛并参与重要生理作用;此外,作用于Wnt和Notch信号通路的药物也存在选择性差的问题。抗器官纤维化药物也可通过调节氧化应激、脂质代谢、MMP抑制剂酶等予以改善。
吡啡尼酮(pirfenidone)和尼达尼布(nintedanib)获得美国FDA审批,用于特发性肺纤维化的治疗。尼达尼布是VEGF、FGF、PDGF等酪氨酸激酶受体的强效抑制剂,吡啡尼酮的作用机制并不十分明确,可能通过抑制TNF–α、IL–6、IL–12、IL–8等炎性介质发挥抗纤维化作用。目前针对器官纤维化的治疗,除了支持治疗、抗炎、干细胞治疗等,尚缺乏有效的药物治疗手段。
发明内容
为解决上述技术问题,本发明提供一种GLYT1抑制剂在治疗器官纤维化中的应用。
本发明采用的技术方案是:GLYT1抑制剂在治疗器官纤维化中的应用。
GLYT1抑制剂在制备治疗器官纤维化药物中的应用。
优选地,通过GLYT1抑制剂抑制甘氨酸转运子GLYT1,减少胶原合成的原料甘氨酸转移到细胞内,从而减少胶原的合成,有效地减少器官纤维化的形成。
优选地,GLYT1抑制剂为RG1678,结构如式1所示;
优选地,给药方式为口服。
优选地,用于肺纤维化。
本发明具有的优点和积极效果是:通过GLYT1抑制剂抑制甘氨酸转运子GLYT1,从而减少胶原合成,可以明显改善博来霉素引起的肺纤维化,对已经形成的肺纤维化也有明显的改善;GLYT1抑制剂已经进入II期临床试验,用于恶性肿瘤的治疗,已有I期临床安全性的数据;不同于靶向TGF-β等通路的治疗手段,该方案对其它生理功能的影响较小。
附图说明
图1实施例1中小鼠体重变化;
图2实施例1中小鼠肺重体重比变化;
图3实施例1中小鼠肺组织石蜡切片HE染色结果;
图4实施例1中小鼠肺组织石蜡切片Masson染色结果;
图5实施例1中小鼠的肺组织石蜡切片Sirius Red染色结果;
图6实施例1中1mg/kg博莱霉素干预后小鼠肺组织切片免疫组化结果;
图7实施例1中5mg/kg博莱霉素干预后小鼠肺组织切片免疫组化结果;
图8实施例2中博莱霉素对A549细胞毒性评估结果;
图9实施例2中GlyT1抑制剂对A549细胞毒性评估结果;
图10实施例2中Western blot法检测肺纤维化相关指标结果;;
图11实施例2中Western blot灰度值分析结果。
具体实施方式
在慢性阻塞性肺疾病中,随着细胞外基质中一型胶原蛋白与III型胶原蛋白的沉积,小气道的弹性逐渐降低,加快疾病恶化的进程。此外,随着肝星状细胞的增生,I型胶原蛋白与III型胶原蛋白逐渐取代IV型胶原蛋白,肝血窦毛细血管的结构逐渐发生病理性改变。血管结构的改变在引发门脉高压和相关疾病的同时,又进一步加重了纤维化的进程。在不同实质器官损伤后,我们都能看到基质中胶原蛋白沉积水平与器官纤维化进程具有强相关性。因此,胶原沉积对于器官纤维化程度判断及预后水平都具有一定的预测功能。
本发明公开一种GLYT1抑制剂再治疗器官纤维化中的应用,GLYT1抑制剂抑制甘氨酸转运子GLYT1,减少胶原合成的原料甘氨酸转移到细胞内,从而减少胶原的合成,有效地减少器官纤维化的形成。本发明某些小鼠实验实施例中,使用的GLYT1抑制剂为RG1678(GlyT1 Inhibitor1),结构如式1所示;
给药剂量为0.79mg/kg*d,给药方式为灌胃,使用时,将药物溶解在含0.3%Tween80的H2O中。
通过GLYT1抑制剂抑制甘氨酸转运子GLYT1,从而减少胶原合成,可以明显改善博来霉素引起的肺纤维化,对其它生理功能的影响较小,用于肺纤维化治疗时,不仅能够对肺纤维化初期有较佳效果,对已经形成的肺纤维化也有明显的改善。GLYT1抑制剂可采用口服方式给药,使用的GLYT1抑制剂已经进入II期临床试验,本是用于恶性肿瘤的治疗;安全性也具有一定保障,用于人体时副作用会较小。
下面结合附图对本发明方案做出说明,其中,未具体说明操作步骤的实验方法,均按照相应商品说明书进行,实施例中所用到的仪器、试剂、耗材如无特殊说明,均可从商业公司购买得到。
实施例1:GLYT1抑制剂RG1678缓解博莱霉素诱导的小鼠肺纤维化
1.1实验动物
C57BL/6J小鼠,雄性,8-10W,体重24-25g,购于北京华阜康生物科技有限公司。小鼠饲养于SPF环境下,室温维持于20-25℃,湿度控制于50-60%,小鼠正常喂食,光照调节为12h昼夜循环模式,造模前小鼠于相应环境中适应性饲养1周。所有动物实验均依从天津医科大学动物护理规定和指南,同时获得天津医科大学动物委员会批准。
1.2实验材料
博莱霉素(BLM,厂家:GLPBIO,美国)、RG1678(GlyT1 Inhibitor1,厂家:Selleck,美国)、生理盐水、阿佛丁(三溴乙醇,厂家:上海麦克林生化科技有限公司,中国)、1ml注射器,261/2G针;眼用软膏、医用镊子、加热灯、手术板的角度约为70°(从水平方向)。
1.3实验方法:(博莱霉素经口滴注小鼠)
1.准备好预稀释的博莱霉素,根据体重给药1mg/kg和5mg/kg。
2.用阿佛丁麻醉小鼠:称量每只小鼠体重,计算阿佛丁剂量(配置浓度1.25%,小鼠剂量:0.2ml/10g);通过抓鼠颈毛抑制小鼠活动,用1mL注射器和261/2G针腹腔注射根据体重计算的剂量。麻醉时间为10-40分钟。
3.在注射麻醉药5分钟内,小鼠会安定下来并停止移动。通过翻正反射消失来验证镇静效果,如果镇静效果达到,则继续下一步。
4.实验过程中应避免小鼠眼睛过于干燥。
5.将所需体积的博莱霉素或无菌PBS装入无菌的200μL吸管头。
6.将鼠标放在手术板上,用手术螺纹环悬吊在上门牙上。确保有足够的光照可以显示声带。
7.用无菌带垫的镊子轻轻拉伸舌头向一侧,向下颌骨方向,以看到声带;然后,将装载博莱霉素的移液管尖放低到口腔后部,吸气时通过声带输送液体。等待听到喘息声,确认是气管内输送液体。对照组动物用等量的无菌PBS代替博莱霉素溶液。
8.松开舌头,小心地将上门牙从悬吊线上取下。将小鼠置于加热灯或垫下,直到麻醉恢复,通常在注射麻醉剂后一小时内。
9.每次使用前后用酒精垫清洁产钳。
10.每天监测小鼠,直到它们被安乐死进行分析。每天根据分组对小鼠进行灌胃RG1678或含0.3% Tween 80的水。
11.实验到达适当节点后,对小鼠进行安乐死处理,摘除小鼠眼球进行取血,后解剖小鼠进行取材。
1.4动物分组
按照上述方法对不同分组小鼠进行造模,对照组采用生理盐水造模,实验组采用不同浓度博莱霉素进行造模,造模14d后,分别采用含0.3% Tween 80的ddH2O或0.3%Tween 80的ddH2O配置的RG1678灌胃处理,连续灌胃14d;具体实验情况如表1所示;
表1
组别 | 造模 | 灌胃 |
对照组1 | 生理盐水 | ddH2O |
实验组1 | 1mg/kg博莱霉素 | ddH2O |
实验组2 | 1mg/kg博莱霉素 | RG1678 |
实验组3 | 5mg/kg博莱霉素 | ddH2O |
实验组4 | 5mg/kg博莱霉素 | RG1678 |
药物配置:
1.10mg/ml博莱霉素储液:称取10mg博莱霉素溶解于1ml的0.9%的氯化钠注射液中。以体重计算每只小鼠滴注量,每只滴注量约50μl,根据不同的分组1mg/kg、5mg/kg分别将储液稀释为0.5mg/ml和2.5mg/ml用于后续滴注。
2.0.3%的Tween 80:将30μl Tween 80与9970μl的ddH2O混合配置成10ml含0.3%的Tween 80的ddH2O。
3.0.79mg/ml的RG1678:取适量RG1678溶于含0.3% Tween 80的ddH2O,配制为0.79mg/ml的工作液储液,后根据小鼠体重计算灌胃量,每只约200μl,使用前需将储液稀释为0.079mg/ml的工作液。
经气道给药后,可发现小鼠立即出现呼吸急促表现,贴近可闻及水泡音。待麻醉苏醒后,BLM组小鼠明显反应迟缓,蜷缩抱团,颤抖,四肢末端出现紫绀现象,随后小鼠饮食减少。体重逐渐降低,皮肤皱缩。BLM+RG1678组小鼠上述改变程度较轻,体重降低水平亦较轻。
于造模前测量小鼠体重,实验过程中每天监测小鼠体重,观察体重变化,并在实验终点与实验初比较。结果如图1-2所示,BLM组较NC组小鼠体重呈现剂量依赖性的显著降低(****P<0.0001),甚至低于其本身初始体重,肺重体重比明显升高(*P<0.05),该升高也呈现剂量依赖性;而BLM+GLYT1 IN组较BLM组体重明显增加、肺重体重比的改变明显减轻(**P<0.01,***P<0.001)。
为进一步细致观察小鼠肺组织损伤情况,本实施例对小鼠肺组织进行病理学检测,HE染色后,于光镜下观察.结果如图3所示,发现NC组组织结构大致正常,肺泡结构清晰。给予BLM刺激后小鼠肺组织结构明显紊乱,肺泡结构消失,肺泡间隔变厚,血管充血明显,可见炎细胞弥漫性渗出、浸润,肺组织实变,纤维化明显。BLM造模后14天开始给予RG1678(GlyT1 Inhibitor1)干预,也能使BLM诱导的肺组织实变减弱,纤维化程度明显减弱。
对小鼠肺组织切片Masson染色,Masson染色法是一种检测组织中胶原纤维分泌及沉积水平的试验方法。经不同染液处理后,组织切片整体背景为红色,细胞核呈黑色,而胶原纤维呈蓝色。根据蓝色深浅及累及范围,可评估胶原纤维的含量。
结果如图4所示,与NC组相比,BLM组小鼠肺组织内可见大量胶原沉积,这种沉积在1mg/kg组及5mg/kg组均十分明显,弥漫性分布,出现明显的肺实变,肺泡结构消失;BLM+GLYT1 IN组的胶原沉积不明显,较BLM组有很大差别,肺泡结构保存良好,无明显的肺实变样改变。可见,在博莱霉素诱导的小鼠肺纤维化模型中,小鼠肺组织胶原被大量分泌,给予GlyT1 Inhibitor1干预可显著减轻博莱霉素诱导的小鼠肺组织胶原沉积。
对小鼠肺组织切片Sirius Red染色。Sirius Red染色法也是一种检测组织中胶原纤维分泌及沉积水平的试验方法。胶原纤维中碱性基团与强酸性天狼星红染液结合,在普通光学显微镜下细胞核呈蓝色,而胶原呈现为红色,在偏振光镜下,出现双折射现象,可将I型和III型胶原纤维进行区分。
如图5所示,结果再次发现NC组小鼠肺组织切片内无明显胶原沉积,肺泡结构相对清晰,在支气管周围可见轻微胶原沉积;给予BLM刺激后小鼠肺组织切片内可见明显的胶原沉积,弥漫性分布;给予GlyT1 Inhibitor1干预后,胶原沉积明显减少,肺泡结构也相对正常清晰,但仍可在支气管周围有轻微胶原沉积,这种干预在造模14天后开始进行便有该效果。
为进一步分析GlyT1 Inhibitor1对肺纤维化造模后胶原蛋白的影响,我们对小鼠肺组织切片进行免疫组化分析,分析其Ⅰ型胶原和Ⅲ型胶原的表达水平。结果如图6-7所示,与NC组相比,BLM干预后,小鼠肺组织内Ⅰ型胶原和Ⅲ型胶原沉积明显,表达水平较高;造模14天后开始给予GlyT1 Inhibitor1进行治疗,14天后,其Ⅰ型胶原和Ⅲ型胶原沉积都较BLM组出现明显减轻,表达水平也出现明显下降。
实施例2:GlyT1 Inhibitor1缓解博莱霉素引起A549细胞破坏及其机制
2.1 A549细胞;
博莱霉素(BLM,厂家:MedChemExpress公司,美国);GlyT1 Inhibitor(GLYT1IN,厂家:MedChemExpress公司,美国);利培酮(RIS,厂家:MedChemExpress公司,美国);COL1A1一抗(爱博泰克生物科技有限公司,中国);COL3A1一抗(爱博泰克生物科技有限公司,中国);COL4A1一抗(爱博泰克生物科技有限公司,中国);β-actin一抗(爱博泰克生物科技有限公司,中国);E-cadherin一抗(爱博泰克生物科技有限公司,中国);α-SMA一抗(爱博泰克生物科技有限公司,中国)。
2.2细胞增殖毒性分析(CCK8)
使用CCK-8试剂盒检测细胞增殖和细胞毒性。其工作原理为:在电子耦合试剂存在的情况下,WST-8(化学名:2-(2-甲氧基-4-硝苯基)-3-(4-硝苯基)-5-(2,4-二磺基苯)-2H-四唑单钠盐),可以被线粒体内的脱氢酶还原生成高度水溶性的橙黄色的甲臜产物,其颜色的深浅与细胞增殖成正比,与细胞毒性成反比,对同样的细胞,颜色的深浅和细胞数目呈线性关系。使用酶标仪在450nm波长处测定OD值,可以间接反映活细胞的数量。
利用CCK8实验检测A549细胞被不同浓度BLM、GlyT1 Inhibitor处理72H后的活性,如图8-9所示,A549细胞的细胞活力随BLM、GlyT1 Inhibitor浓度的增加而降低,10μM的博莱霉素刺激便可引起统计学差异。本实施例选用A549细胞活力为80%左右时的BLM进行刺激,以25μM的BLM对A549细胞进行刺激造模;随着GLYT1 IN浓度的升高,A549细胞的活性逐渐降低。在380nM条件下,A549细胞的活性大于50%,为更易获取稳定的实验结果,后续研究中选择190nM的GLYT1 IN作为本研究处理浓度。
2.3博莱霉素破坏作用下GlyT1 Inhibitor对肺泡上皮细胞的影响
在体外构建博莱霉素诱导的肺纤维化模型,并同时应用GLYT1抑制剂进行干预。
在体外构建博莱霉素诱导的肺纤维化模型,对A549细胞分为四个处理组:NC组(对照组:给予生理盐水处理)、BLM组(博莱霉素处理组:给予25μM的BLM处理72h)和BLM+GLYT1IN组(博莱霉素和甘氨酸转运子1抑制剂处理组:给予25μM的BLM处理72h,190nM的GLYT1 IN处理72h)。
采用Western blot法检测肺纤维化相关指标col1α1、col3α1、col4α1、E-cadherin(E-Cad)、α-SMA蛋白表达水平。结果如图10-11所示,在BLM刺激后,col1α1、col3α1、col4α1、α-SMA蛋白表达水平显著升高,E-cadherin蛋白表达水平显著下降,添加GlyT1 Inhibitor干预组相较BLM组,col1α1、col3α1、col4α1、α-SMA蛋白表达水平显著下降,E-cadherin蛋白表达水平显著上升。结果显示,GlyT1抑制剂,确实能改善由博莱霉素诱导的细胞水平上的变化,提示其对肺纤维化确有改善效果。
由以上细胞及动物模型的研究结果可以看出,GlyT1抑制剂1可以显著改善器官纤维化。
以上对本发明的实施例进行了详细说明,但所述内容仅为本发明的较佳实施例,不能被认为用于限定本发明的实施范围。凡依本发明申请范围所作的均等变化与改进等,均应仍归属于本发明的专利涵盖范围之内。
Claims (6)
1.GLYT1抑制剂在治疗器官纤维化中的应用。
2.GLYT1抑制剂在制备治疗器官纤维化药物中的应用。
3.根据权利要求1或2所述的应用,其特征在于:通过GLYT1抑制剂抑制甘氨酸转运子GLYT1,减少胶原合成的原料甘氨酸转移到细胞内,从而减少胶原的合成,有效地减少器官纤维化的形成。
4.根据权利要求1或2所述的应用,其特征在于:GLYT1抑制剂为RG1678,结构如式1所示;
5.根据权利要求4所述的应用,其特征在于:给药方式为口服。
6.根据权利要求1或2所述的应用,其特征在于:用于肺纤维化。
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