CN113827655B - 重楼皂苷ⅵ在制备预防和/或治疗肺纤维化的药物中的用途 - Google Patents
重楼皂苷ⅵ在制备预防和/或治疗肺纤维化的药物中的用途 Download PDFInfo
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- CN113827655B CN113827655B CN202111302313.9A CN202111302313A CN113827655B CN 113827655 B CN113827655 B CN 113827655B CN 202111302313 A CN202111302313 A CN 202111302313A CN 113827655 B CN113827655 B CN 113827655B
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Abstract
本发明提供了重楼及其提取物在制备预防和/或治疗肺纤维化的药物中的用途。本发明通过建立体内外肺纤维化评价模型证实了重楼皂苷Ⅵ具有降低博来霉素诱导的肺纤维化小鼠肺组织中α‑SMA、CollagenⅠ表达水平,升高E‑cadherin表达水平,以及抑制肺组织胶原蛋白增生的作用,故重楼皂苷Ⅵ或其盐可用于制备防治肺纤维化的药物,是一种具有巨大临床应用潜力,可用于肺部疾病治疗的天然化合物。
Description
技术领域
本发明涉及医药技术领域,具体涉及重楼皂苷Ⅵ在防治肺纤维化的药物中的用途。
背景技术
肺纤维化(pulmonary fibrosis,PF),即肺脏间质组织由胶原蛋白、弹性素及蛋白醣类构成,当成纤维细胞受到化学性或物理性伤害时,会分泌胶原蛋白进行肺间质组织的修补,进而造成肺脏纤维化;即肺脏受到伤害后,人体修复产生的结果。PF是一种慢性、进行性和纤维化性的间质性肺炎,可分为特发性和继发性,发病率在欧洲和北美较高(每10万人2.8~18例),在亚洲和南美较低(每10万人0.5~4.2例,可能需要更新数据),而死亡率高于大多数肿瘤,被称为一种“类肿瘤疾病”。PF在男性中更为常见,且好发于中老年人群,中位生存时间为3~5年,且5年的生存率小于30%,具有慢性、进行性、不可逆性、致死性等特征,目前除肺移植外尚无治愈的方法。PF作为纤维化性间质性肺疾病,是一种临床较为常见的呼吸系统疾病,病变主要发生在机体肺组织,主要临床表现为呼吸困难、咳嗽、咳痰、Velcro啰音等,对患者的身体健康和生活质量造成严重的影响。
PF的发病机制尚不十分清楚,与慢性炎症、基因易感等多因素有关,涉及激活许多促纤维化炎症因子和细胞类型,诱导细胞外基质(extracellular matrix,ECM)的过量产生从而导致组织纤维化。越来越多的证据表明,肺泡上皮细胞凋亡可能是该病的初始触发因素,遗传背景和环境暴露均可能导致这一结果,并启动了肺泡上皮细胞凋亡“衰老相关分泌途径”最终导致全身性疾病,但并非所有处于这种环境下的人都一定会发展出与临床相关的疾病,这还要取决于接触这些因素的程度和持续时间。在疾病的起始阶段,上皮细胞功能障碍的分子介质如内质网应激、TGF-β过度激活、生长因子,趋化因子分泌会导致EMT、纤维细胞募集和成纤维细胞分化。这些过程使得疾病进入进展阶段,在此阶段病理性间充质细胞释放出异常类型和数量的基质蛋白,这些基质蛋白导致了肺的重塑。另外成纤维细胞内病理改变的基质或表观遗传变化可能导致间充质细胞活化,也可能是进行性纤维化的前馈通路。
研究发现多种促纤维化途径促进PF发生发展,虽然目前已完成或正在进行多项针对性药物开发,但仍没有一种可以完全治愈的药物。美国食品药品管理局已批准的治疗PF的药物吡非尼酮和尼达尼布均被认为可以延缓PF进程,包括延缓肺功能及运动耐力的下降、延长生存期等。然而,吡非尼酮及尼达尼布应用过程中均存在轻度至中度不良事件,吡非尼酮主要表现为胃肠道症状(恶心、消化不良)、肝功能异常和皮肤光过敏;尼达尼布表现为胃肠道不良反应(腹泻、恶心和呕吐)和肝酶水平增高,这些药物可以减缓疾病恶化,但并不能治愈肺纤维化。且肺纤维化是一种愈后极差的难治性疾病,其形成病因及发病机制尚不清楚,且其病情呈不可逆发展的趋势。
中药具有悠久的历史,近年来对于肺纤维化的疗效频频得到验证,以其多途径、多靶点、副作用少、疗效显著成为当下治疗PF替代疗法之一。但目前有关中医药疗法治疗肺纤维化的研究仍存在一定的问题,如大量实验只进行对病治疗,缺乏对证治疗的研究;研究多以中药复方为主,其组成的多样性致使研究靶点、机制多重交叉,难以辨识出引起作用机制的有效成分,致使无法揭示其药理作用,更无法使其治疗效果得到科学的论证和体现。
中药重楼药用历史悠久,以蚤休之名首载于《神农本草经》,在《唐本草》中录别名为重楼,以后历代本草古籍都有论述,均以根茎人药,其基源为重楼属多种植物。1995年版中国药典收载有云南重楼Parispolyphylla var.yunnanensis和七叶一枝花P.polyphyllavat.chinensis两个变种。本属植物主要有清热解毒、消肿止痛、凉肝定惊等功效,用于疔肿痈肿、咽喉肿痛、毒蛇咬伤、跌打伤痛、惊风抽搐等证,是著名中成药云南白药、季德胜蛇药片等的主要组成药物。近年来药理研究表明该属植物具有止血、抗肿瘤、抗生育、免疫调节及心血管等多方面的生理活性。专利CN 106668041A公开了重楼中重楼皂苷Ⅵ具有治疗肺癌的作用,但肺癌与肺纤维化属于不同的疾病,病理表现并不相同,目前还没有关于重楼皂苷Ⅵ对肺纤维化作用的报道。
发明内容
为解决上述问题,本发明提供了重楼及其提取物在制备预防和/或治疗肺纤维化的药物中的用途
进一步地,所述提取物为重楼皂苷。
更进一步地,所述重楼皂苷包括重楼皂苷I、重楼皂苷II、重楼皂苷V、重楼皂苷Ⅵ、重楼皂苷ⅥI、重楼皂苷D。
更进一步地,所述重楼皂苷为重楼皂苷Ⅵ及其药学上可接受的盐。
进一步地,所述药物是预防和/或治疗特发性肺纤维化的药物。
进一步地,所述药物是预防和/或治疗继发性肺纤维化的药物;所述继发性肺纤维化是粉尘、化学气体、病原体、药物、放化疗或疾病累及诱导的肺纤维化。
更进一步地,所述药物是预防和/或治疗博来霉素诱导的肺纤维化。
进一步地,所述药物是逆转肺组织结构损伤和/或抑制肺组织胶原蛋白增生的药物。
进一步地,所述药物是降低博来霉素诱导的肺纤维化小鼠肺组织中α-SMA、CollagenⅠ表达水平,和/或升高E-cadherin表达水平的药物。
本发明还提供了一种预防和/或治疗肺纤维化的组合物,它是以重楼皂苷Ⅵ和/或其药学上可接受的盐为活性成分,加入药学上可接受的辅料或者辅助性成分制备成的口服制剂。
本发明重楼皂苷Ⅵ在制备预防和/或治疗肺纤维化的药物中的用途,通过建立体内外肺纤维化评价模型证实了重楼皂苷Ⅵ具有降低博来霉素诱导的肺纤维化小鼠肺组织中α-SMA、CollagenⅠ表达水平,升高E-cadherin表达水平,以及抑制肺组织胶原蛋白增生的作用,基于此,重楼皂苷Ⅵ或其盐可用于制备防治肺纤维化的药物,是一种具有巨大临床应用潜力,可用于肺部疾病治疗的天然化合物。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1.重楼皂苷Ⅵ抑制各细胞增殖分析图(A:A549;B:NIH/3T3;C:HPF;D:QRPLF;E:ARPLF;F:重楼皂苷Ⅵ对各细胞作用24h的IC50)
图2.重楼皂苷Ⅵ对TGF-β1诱导的上皮细胞A549的形态显微图以及蛋白电泳图(A:A549细胞划痕实验;B:划痕愈合率%;C:A549细胞形态图;D:A549细胞蛋白电泳图)
图3.重楼皂苷Ⅵ对NIH/3T3增殖影响以及菌落统计分析(A:NIH/3T3细胞的克隆实验;B:NIH/3T3细胞菌落统计图)
图4.重楼皂苷Ⅵ对TGF-β1诱导NIH/3T3中促纤维化相关蛋白的电泳图及免疫荧光图(A:蛋白电泳图;B:免疫荧光图)
图5.在预防给药实验中,重楼皂苷Ⅵ抑制博来霉素诱导的肺纤维化小鼠肺组织中羟脯氨酸含量以及肺组织结构的改变(A:重楼皂苷Ⅵ预防BLM所致肺纤维化的实验方案;B:肺组织中羟脯氨酸的含量;C:重楼皂苷Ⅵ减轻BLM所致肺纤维化小鼠模型的H&E染色和Masson染色)
图6.在预防给药实验中,重楼皂苷Ⅵ抑制博来霉素诱导的肺纤维化小鼠肺组织中纤维化相关蛋白表达(A:免疫组化检测肺组织CollagenⅠ、α-SMA表达;B蛋白免疫技术检测肺组织中CollagenⅠ、α-SMA和E-cadherin的表达)
图7.在治疗给药实验中,重楼皂苷Ⅵ逆转了博来霉素诱导的小鼠的纤维化(A:重楼皂苷Ⅵ逆转BLM诱导的肺纤维化模型的给药示意图;B:肺组织中羟脯氨酸的含量;C:肺重系数分析;D:小鼠体重变化;E:肺组织切片的H&E染色和Masson染色)
图8.在治疗给药实验中,重楼皂Ⅵ抑制博来霉素诱导的肺纤维化小鼠肺组织中纤维化相关蛋白表达(A:免疫组化检测肺组织CollagenⅠ、α-SMA表达;B蛋白免疫技术检测肺组织中CollagenⅠ、α-SMA、E-cadherin和Vimentin的表达)
具体实施方式
本发明具体实施方式中使用的原料、设备均为已知产品,通过购买市售产品获得。
实施例1重楼皂苷Ⅵ抑制肺纤维化的体外细胞实验
NIH/3T3(小鼠胚胎成纤维细胞)、A549(人肺泡腺癌基底上皮细胞)、HPF(人肺成纤维细胞)、鼠源肺成纤维细胞的过度增殖会造成胶原的沉积,所以,抑制它们的表达能抑制胶原沉积;A549被TGF-β1(转化生长因子β1蛋白)刺激后会加速它的EMT转化(上皮细胞-间充质转化),进而加速肺纤维化的形成;NIH/3T3、HPF被TGF-β1刺激后会激活,转化为肌成纤维细胞,分泌更多的胶原蛋白,进而加速肺纤维化的形成。所以,抑制EMT转化能降低肺纤维化的形成。所以,通过如下试验探究重楼皂苷Ⅵ对肺纤维化的影响。
1.细胞的培养
NIH/3T3细胞用含10%FBS DMEM高糖培养基培养;A549用10%FBS的10%FBS DMEM培养基培养;HPF用10%FBS DMEM培养基培养;鼠源肺成纤维细胞用10%FBS的F-12培养基培养。以上细胞系在培养和实验过程中均需要加入100U/mL的青霉素和链霉素,培养在环境条件为37℃、5%CO2的孵箱中。
2.鼠源肺成纤维细胞的提取
从达硕购买的6只SD大鼠(180-220g,8周龄,雄性),其中3只用于造模提取激活态大鼠原代肺成纤维细胞(activated rat primary lungfibroblasts,ARPLF),3只用于提取静息态大鼠原代肺成纤维细胞(quiescent rat primary lung fibroblasts,QRPLF)。在SPF级的条件下适应性培养一周后,采用10%的水合氯醛腹腔注射麻醉大鼠。将麻醉后的大鼠固定在干净的泡沫板上,先用酒精棉擦拭大鼠颈部,然后用无菌手术剪沿大鼠下颚往下剪开其颈部皮肤,镊子剥离找到支气管,用1mL注射器气管内给予博来霉素(5mg/kg),双手提起大鼠前肢,上下左右均匀摇晃,使博来霉素均匀分布在其肺部。将大鼠颈部皮肤用缝合线缝合,并擦拭碘伏。造模2周后提取肺成纤维细胞。先将大鼠麻醉(10%的水合氯醛),后通过腹主动脉取血后取出整个肺,用Hanks液清洗,去除掉筋膜等。将肺剪碎,用含有EDTA的胰蛋白酶消化45min。取出用70μM的筛网过筛后取上清,1500r/5min离心,去上清加入培养基混匀后500r/5min离心。取上清后1500r/min离心,去上清,得到的即为ARPLF。QRPLF的提取步骤和ARPLF相同,只是不需要进行造模,取第三代到第八代进行试验。
3.使用CCK-8试剂盒检测重楼皂苷Ⅵ对5种细胞增殖的影响
选取对数期生长状态良好的5种细胞,根据细胞生长速度接种96孔板,每孔约1500-2000个细胞,每孔100μL,在37℃、5%CO2培养箱培养。接种第二天,每孔加入100μL用相应培养基稀释过的重楼皂苷Ⅵ,使其终浓度为0μg/mL、0.625μg/mL、1.25μg/mL、2.5μg/mL、5μg/mL、10μg/mL、20μg/mL,相同浓度设置5个复孔,于37℃、5%CO2培养箱分别培养24、48、72h后,每孔加入10μL CCK-8试剂并置于孵箱中孵育2-4小时。酶标仪于450nm处测定OD值。实验重复三次,整理数据,根据吸光度值得出的生长抑制率与重楼皂苷Ⅵ浓度,计算出IC50。
实验结果见图1A、1B、1C、1D、1E,分别可以看出PPⅥ能抑制A549、NIH/3T3、HPF、QRPLF、ARPLF细胞增殖。
4.检测重楼皂苷Ⅵ对TGF-β1刺激后A549的迁移的影响
将处于对数生长期的A549细胞用胰酶消化,以105个/孔接种到6孔板里。待细胞生长至80%后,用不含血清的培养基饥饿6h,用200μL的枪尖划痕,用生理盐水洗两次,换成完全培养基,用倒置显微镜拍摄0h的划痕宽度,并记录拍摄位置。然后继续用5ng/mL TGF-β1刺激,1h后加入重楼皂苷Ⅵ(5μM、10μM),于37℃、5%CO2培养箱培养24h后拍摄与0h同一位置的划痕宽度。然后用Image J统计划痕的愈合率。
结合图2A和图2B可以看出,与对照相比,TGF-β1处理后A549迁移能力增强。与单独使用TGF-β1相比,重楼皂苷Ⅵ与TGF-β1的联合处理抑制了A549细胞迁移,由此可见,重楼皂苷Ⅵ能削弱TGF-β1诱导的上皮细胞的迁移。
5.检测重楼皂苷Ⅵ对TGF-β1刺激后A549的形态变化的影响
选取对数生长期的细胞,消化收集细胞,离心弃上清,加入新鲜培养基后轻轻吹打混匀,细胞计数并均匀接种到铺有玻片的6孔板中,每孔2mL细胞悬液,细胞密度以接种后48h对照孔能长满。接种24h后,用无血清的培养基饥饿6h,加入含有5ng/mL TGF-β1的完全新鲜培养基刺激,1h后加入5μM、10μM重楼皂苷Ⅵ继续放入培养箱培养。加药24h后,取出6孔板,用倒置显微镜白光观察细胞的形态变化。
从图2C可以看出,用TGF-β1刺激A549,诱导A549发生EMT,观察到A549细胞在TGF-β1诱导下由卵形上皮细胞向纺锤形向成纤维细胞的形态转变,重楼皂苷Ⅵ的处理抑制了这种转变。由此可见,重楼皂苷Ⅵ能抑制TGF-β1刺激诱导的A549形态的变化。
6.检测重楼皂苷Ⅵ对TGF-β1刺激后A549的EMT转化蛋白表达的影响
取对数生长期的细胞,进行细胞传代,传代的细胞密度以对照组48h后长到80%-90%为宜,分为4皿。24h后,用无血清的培养基饥饿6h,加入含有5ng/mL TGF-β1的完全新鲜培养基刺激,1h后加入5μM、10μM重楼皂苷Ⅵ继续放入培养箱培养。加药24h后,取出处理的培养皿,放置在冰上10min。弃去上清液,用PBS轻轻洗两遍,留1mL PBS,用细胞刮片收集皿中的细胞,4℃3000rpm/min离心3min,尽量将上清液吸干净,加入适量的RIPA裂解液,用移液枪吹打数下,使裂解液与细胞充分接触,裂解细胞,每隔10min涡旋一次,涡旋5-6次,并用超声波细胞粉碎仪超声2-3遍。细胞充分裂解后,4℃,13000rpm/min离心3-5min,取上清,用Bradford法进行蛋白定量,加入5×SDS上样缓冲液,加热5-10min使蛋白变性,放入-20℃保存。按照SDS-PAGE凝胶试剂盒的步骤,配制7.5%、10%或12.5%的凝胶,凝胶浓度根据目的蛋白的大小决定。每孔上样量均为50μg,80V电泳30min左右,当样品进入分离胶后转为100V电泳60min左右,当样品跑到分离胶底部时可停止电泳。电泳结束后,将与分离胶大小相当的PVDF膜放入甲醇中浸泡10s左右,之后将膜放入转膜缓冲液中。将凝胶和PVDF膜按照(红面)海绵→3张滤纸→PVDF膜→凝胶→3张滤纸→海绵(黑面)的顺序制成转膜“三明治”结构,排除各层之间的气泡,并迅速插入转膜夹中,电压100V进行转膜,转膜时间由目的蛋白分子量大小决定。转膜结束后,将膜放入封闭缓冲液中,室温封闭1h。封闭过后的膜用TBST洗膜液洗五分钟,然后根据抗体说明书用一抗稀释液将一抗稀释至合适的浓度,把膜放入稀释过后的一抗当中,4℃过夜。之后将膜取出置于水平摇床上,用TBST洗膜液洗3次,每次5min,用封闭液稀释二抗,37℃孵育膜1h。取出膜,TBST中洗3-4次,每次十分钟,将显影液均匀滴加到PVDF膜上,在曝光仪中曝光。
从图2D可以看出,TGF-β1处理后α-SMA和CollagenⅠ的蛋白表达升高,以及E-cadherin的蛋白表达降低,重楼皂苷Ⅵ减轻了这些变化。由此可见,重楼皂苷Ⅵ能减轻TGF-β1诱导的A549的EMT。
7.检测重楼皂苷Ⅵ对NIH/3T3的增殖的影响
集落形成试验:将NIH/3T3细胞以指定数量(400–600个细胞/孔)接种在六孔板中。孵育24h后,将细胞用不同浓度的PPⅥ处理,然后再孵育12天。然后用甲醇固定细胞,用0.5%结晶紫溶液染色15min,显微镜下计数菌落数(>50个细胞)。
从图3A、3B可以看出,随重楼皂苷Ⅵ浓度增加,NIH/3T3细胞菌落数逐渐减少,当重楼皂苷Ⅵ浓度达到10μM时,从显微镜下已观察不到NIH/3T3细胞,由此可见,重楼皂苷Ⅵ能削弱NIH/3T3细胞的增殖能力。
8.检测重楼皂苷Ⅵ对NIH/3T3细胞纤维化相关蛋白表达变化的影响
细胞的Western blot检测相关蛋白:取对数生长期的细胞,进行细胞传代,传代的NIH/3T3细胞密度以对照组48h后长到80%-90%为宜,分为4皿。24h后,用无血清的培养基饥饿6h,加入含有5ng/mL TGF-β1的完全新鲜培养基刺激,1h后加入10μM重楼皂苷Ⅵ继续放入培养箱培养。后续提取蛋白的步骤与上述相同。
从图4A可以看出,重楼皂苷Ⅵ降低了由TGF-β1诱导的与纤维化相关的α-SMA和CollagenⅠ蛋白的表达,由此可见,重楼皂苷Ⅵ能抑制3T3细胞的活化。
免疫荧光实验:1×104NIH/3T3细胞/孔在预包被的细胞载玻片上的24孔板中培养24h,然后,将培养基替换为无血清培养基,在大约6h的饥饿条件下进行。接下来,将与5ng/mL TGFβ-1混合的DMEM完全培养基添加到饥饿的细胞中,1小时后,添加10μM重楼皂苷Ⅵ。24h后,将细胞在4%多聚甲醛中于25℃固定10min,并用0.5%Triton X-100PBS透化10min。用封闭缓冲液(含0.05%Triton X-100和5%牛血清白蛋白的PBS)处理后,将细胞与抗α-SMA(1:200;Abcam,Cambridge,MA)和抗E-cadherin(1:500;Abcam,剑桥,马萨诸塞州)在4℃过夜。将细胞洗涤3次,并与FITC偶联的山羊抗小鼠/兔抗体(1:500,Alexa Fluor 488;LifeTechnologies,Waltham,MA)在室温避光孵育2h。细胞核用DAPI(Roche MolecularBiochemicals,Inc.,Pleasanton,CA)染色10min。在荧光显微镜(Nikon 80i,Tokyo,Japan)下分析免疫荧光。
从图4B可以看出,重楼皂苷Ⅵ降低了由TGF-β1诱导的与纤维化相关的α-SMA和CollagenⅠ蛋白的表达,由此可见,重楼皂苷Ⅵ能抑制3T3细胞的活化。
实施例2重楼皂苷Ⅵ治疗博来霉素诱导的肺纤维化的体内动物实验
在支气管滴注博来霉素(BLM)后,BLM可诱导肺泡Ⅰ型细胞凋亡并抑制肺泡Ⅱ型细胞转化为Ⅰ型细胞,阻止其对肺泡结构的修复。它也诱导成肌纤维细胞的异常增生,过量胶原纤维堆积,最终导致肺纤维化的形成。与人类弥漫性肺纤维化或纤维化肺泡炎相似的变化的一致诱导表明,博来霉素诱导的损伤可以为研究这种不明确的疾病组提供合适的模型。在众多品系中C57BL/6小鼠对BLM的敏感性相对较高,易被诱导形成肺纤维化,且肺脏组织学特征与人类相似,更能反映临床上IPF的发展机制和病理特征,故认为C57BL/6小鼠是小鼠肺纤维化模型的最优选择之一。基于此,建立C57BL/6小鼠博来霉素诱导肺纤维化模型评价重楼皂苷Ⅵ在体内治疗肺纤维化的新用途。
(1)小鼠肺纤维化模型的建立
6-8周龄雄性C57BL/6小鼠。用5mg/mL戊巴比妥钠腹腔注射麻醉小鼠。麻醉成功后,用无菌器械剪开其颈部皮肤,暴露出支气管。找到其支气管后,用注射器注入2mg/kg/10mL博来霉素,缓慢注射所有博来霉素后再缓慢拔出针尖,然后双手提起小鼠前肢,上下左右均匀摇晃,使博莱霉素均匀分布在其肺部。然后用手术缝合线将剪开的颈部皮肤缝合,并喷以碘伏消毒。造模完成后,将小鼠平放于干净笼具内复苏,注意保暖,观察小鼠心跳、呼吸情况,防止窒息。
(2)重楼皂苷Ⅵ预防博来霉素诱导的肺纤维化实验
在博来霉素支气管滴注(2mg/kg)的第1天,将所有小鼠随机分为4组。假手术组每天腹腔注射生理盐水。溶剂组腹腔注射溶剂对照组。重楼皂苷Ⅵ给药组接受低剂量重楼皂苷Ⅵ(2.5mg/kg)或者高剂量重楼皂苷Ⅵ(5mg/kg)腹腔注射给药。每天腹腔注射给药一次,共给药20天。第21天处死小鼠。给药期间每3天称重,同时观察动物的健康状况,并记录动物的死亡情况。实验方案如图5A所示。
(3)重楼皂苷Ⅵ治疗博来霉素诱导的肺纤维化实验
在博来霉素支气管滴注(2mg/kg)的第1天,观察小鼠的健康状态,规律饲养1周。在第7天时,将所有小鼠随机分为4组,假手术组每天腹腔注射生理盐水。溶剂组腹腔注射溶剂对照组。重楼皂苷Ⅵ给药组接受高剂量重楼皂苷Ⅵ(5mg/kg)腹腔注射给药。每天腹腔注射给药一次,共给药14天。第21天处死小鼠。给药期间每3天称重,同时观察动物的健康状况,并记录动物的死亡情况。实验方案如图7A所示。
1.重楼皂苷Ⅵ对博来霉素肺纤维化小鼠的肺重系数、肺组织羟脯氨酸含量、体重的影响
体内模型给药结束后,取小鼠肺组织于-80℃存放。取肺组织,解冻,精确称重30mg以上。剪碎后,加入碱性水解液,沸水浴20min水解。水解完成后根据试剂盒里的指示剂等调PH为6.0-6.8,然后用双蒸水定容至10mL。取4mL稀释后的水解液于BD管里,加入20mg-30mg左右的活性炭,混匀后离心,3500r/10min,小心取上清1mL用于检测。根据羟脯氨酸检测试剂盒制造商的说明书,依次加入相关试剂一、二、三,最后60℃水浴15min,冷却后,3500r离心10min,取上清用分光光度计测量其在550nm波长,1cm光径下的吸光度值。再根据如下公式计算:
从图5B、图7B可以看出,重楼皂苷Ⅵ能降低博来霉素诱导肺组织羟脯氨酸含量的升高。此外从图7C可以看出,重楼皂苷Ⅵ能抑制博来霉素诱导的肺重系数的增加;从图7D可以看出,重楼皂苷Ⅵ能减轻博来霉素诱导的小鼠体重的降低。由此可见,重楼皂苷Ⅵ能预防甚至逆转BLM诱导的肺纤维化。
2.重楼皂苷Ⅵ对博来霉素诱导的肺组织结构改变(H&E染色)和胶原增生(Masson染色)的影响
H&E染色:先用苏木精染色2min,然后用流动的自来水冲洗至水里无色,注意不能直接用水冲洗玻片;接着用1%的盐酸酒精水化10s,洗去细胞质中残留的苏木精,至只有细胞核着色,再用自来水清洗残留的盐酸酒精1min;接下来伊红染色10s,然后自来水稍洗后脱水。85%酒精、95%酒精、无水乙醇中各3min,最后再依次浸入二甲苯Ⅰ,二甲苯Ⅱ各10min。拿出玻片置于通风橱晾干后用中性树胶封片,待中性树胶晾干后用正置显微镜拍摄。
Masson染色:先用免疫组化笔将组织圈出,滴加适量的苏木素,以覆盖组织为宜,染色8min,然后用蒸馏水洗至不再脱色,接着用1%的盐酸酒精分化至细胞质无色(在镜下观察),再用酸性品红染色90min,在自来水里稍洗之后用磷钼酸分化,然后滴加苯胺蓝,染色3min,最后用1%冰醋酸洗至不再脱色后同H&E染色一样进行脱水,封片。通过正置显微镜拍摄,利用Image J统计胶原容积分数。
从图5C、7E可以看出,与对照组相比,BLM处理后小鼠的肺泡间隙缩小且肺泡间隔增厚,且增强了肺部炎症反应,破坏肺泡结构。由此可见,重楼皂苷Ⅵ能预防和治疗逆转博来霉素诱导的肺组织结构损伤。
3.重楼皂苷Ⅵ对博来霉素诱导的肺纤维化小鼠模型中纤维化相关蛋白变化的影响
免疫组化:先用免疫组化笔将组织圈出,滴加适量的H2O2,以覆盖组织为宜,室温避光10min后用纯水清洗2次,各5min。接着进行抗原修复,将切片置于抗原修复液中,以淹没切片为宜,置于高压锅内加热,待高压锅上汽后3min停止加热,待冷却置室温。取出切片,用无K+PBS清洗3次,每次5min。通过吸水纸吸去背面和组织周围的液体后,用胎牛血清:PBS=1:20的封闭液封闭,37℃20min,保持切片湿润。甩去血清后直接滴加一抗,4℃孵育过夜。第二天回收一抗后,用无K+PBS清洗3次,每次5min(摇床,慢速)。然后滴加二抗,在37℃下孵育20min,随后用PBS缓冲液洗3次,每次5min;使用DAB显色试剂盒显色,约2min后用苏木素染色2min;用自来水清洗5min后对玻片按照H&E染色的过程脱水,使用中性树胶封片;最后在显微镜下观察、拍照。
从图6A、图8A的免疫组化图可以看出,重楼皂苷Ⅵ的治疗逆转了博来霉素小鼠肺组织中α-SMA和CollagenⅠ表达的升高,由此可见,重楼皂苷Ⅵ可预防和逆转博来霉素诱导的肺纤维化。
组织的Western blot检测相关蛋白:将肺组织放入组织研磨仪中研磨破碎。将收集到的组织样品在适当体积的RIPA裂解液中裂解,将组织悬液至于冰上,每隔10min涡旋混匀,并用超声破碎机超声使其充分裂解。细胞充分裂解后,4℃,13000rpm/min离心3-5min,取上清,用Bradford法进行蛋白定量,加入5×SDS上样缓冲液,加热5-10min使蛋白变性,放入-20℃保存。后续提取蛋白的步骤与上述相同。
从图6B、图8B的蛋白印迹图可以看出,重楼皂苷Ⅵ的治疗减轻了BLM诱导的E-cadherin、Vimentin、α-SMA和CollagenⅠ表达的变化,由此可见重楼皂苷Ⅵ可预防和逆转博来霉素诱导的肺纤维化。
综上,本发明通过建立体内外肺纤维化评价模型证实了重楼皂苷Ⅵ具有降低肺组织中α-SMA、CollagenⅠ表达水平,升高E-cadherin表达水平,以及抑制肺组织胶原蛋白增生的作用,基于此,重楼皂苷Ⅵ或其盐可用于制备防治肺纤维化的药物,是一种具有巨大临床应用潜力,可用于肺部疾病治疗的天然化合物。
Claims (6)
1.重楼皂苷Ⅵ作为唯一活性成分在制备预防和/或治疗肺纤维化的药物中的用途。
2.根据权利要求1所述的用途,其特征在于:所述药物是预防和/或治疗特发性肺纤维化的药物。
3.根据权利要求1所述的用途,其特征在于:所述药物是预防和/或治疗继发性肺纤维化的药物;所述继发性肺纤维化是粉尘、化学气体、病原体、药物、放化疗或疾病累及诱导的肺纤维化。
4.根据权利要求3所述的用途,其特征在于:所述药物是预防和/或治疗博来霉素诱导的肺纤维化。
5.根据权利要求1所述的用途,其特征在于:所述药物是逆转博来霉素诱导的肺纤维化小鼠肺组织结构损伤和/或抑制肺组织胶原蛋白增生的药物。
6.根据权利要求1所述的用途,其特征在于:所述药物是降低博来霉素诱导的肺纤维化小鼠肺组织中α-SMA、 CollagenⅠ表达水平,和/或升高E-cadherin表达水平的药物。
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