CN103622983B - 续断皂苷x在制备预防或治疗肺纤维化的药物中的应用 - Google Patents
续断皂苷x在制备预防或治疗肺纤维化的药物中的应用 Download PDFInfo
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Abstract
本发明提供了续断皂苷X的药物新用途，具体涉及续断皂苷X通过抑制上皮细胞间质转化和成纤维细胞增殖从而抑制肺纤维化的发生发展，达到预防或治疗肺纤维化。在上述应用中，续断皂苷X使用剂量范围是50mg～2000mg；优选50～1000mg。
Description
技术领域
本发明涉及续断皂苷X抑制肺纤维化的发生发展从而预防或治疗肺纤维化；具体涉及续断皂苷X通过抑制上皮细胞-间质转化(EMT)和抑制肺成纤维细胞异常活化从而抑制肺纤维化的发生发展，达到预防或治疗肺纤维化。
背景技术：
特发性肺纤维化(idiopathicpulmonaryfibrosis，IPF)又称为隐源性致纤维化性肺泡炎(cryptogenicfibrosingalveolitis，CFA)，是一种慢性炎症性间质性疾病，以普通型间质性肺炎(usualinterstitialpneumonia，UIP)为特征性病理改变且病因不明。主要表现为成纤维细胞灶(fibroblasticfoci)的出现致大量细胞外基质(extracellularmatrix，ECM)沉，胶原积聚，肺泡结构破坏，最终导致正常肺组织结构的破坏，是一种慢性、进行性、不可逆转也是最常见的一种致命性肺疾病[KingTJ，PardoA，SelmanM.Idiopathicpulmonaryfibrosis[J].Lancet.2011，378(9807)：1949-1961.]。临床表现为进行性呼吸困难并伴有刺激性干咳，肺功能限制性通气障碍，病情持续恶化，最终因呼吸衰竭而死亡。流行病学研究显示IPF发病率呈不断上升趋势，目前约为16.3/100,000，且3年内急性恶化发生率为20.7％[NavaratnamV，FlemingKM，WestJetal.TherisingincidenceofidiopathicpulmonaryfibrosisintheUKJ.Thorax，2011，66(6)：462-467.；SongJW，HongSB，LimCMetal.Acuteexacerbationofidiopathicpulmonaryfibrosis：incidence，riskfactorsandoutcome[J].EurRespirJ，2011，37(2)：356-363.]。肺纤维化主要是肺组织受损后修复调节失控、重建异常所引起的病变，期间一系列细胞因子和生长因子等表达异常、炎症反应参与肺纤维化的发病过程。由此造成上皮细胞缺损、成纤维细胞增生和ECM积聚等主要病变，最终结果是成纤维细胞替代了行使正常功能的肺泡上皮细胞，导致了纤维化的发生。肺泡上皮细胞(AECs)的上皮细胞-间质转化(epithelial-mesenchymaltransition，EMT)是一种完全分化的肺泡上皮细胞经历细胞表型改变转化成完全分化的间质细胞的过程，通常转化成肺成纤维细胞和肌纤维母细胞，与肺纤维化密切相关[GuarinoM，TosoniA，NebuloniM.Directcontributionofepitheliumtoorganfibrosis：epithelial-mesenchymaltransition.HumPathol，2009，40(10)：1365-1376.].成纤维细胞异常活化分化成肌成纤维细胞，是肺纤维化合成大量的细胞外基质(ECM)的来源(CowardWR，SainiG，JenkinsG.Thepathogenesisofidiopathicpulmonaryfibrosis.TherAdvRespirDis.2010；4(6)：367-388.)。IPF瘢痕肌成纤维细胞参与失调伤口愈合反应，导致肺的结构扭曲和功能失调，破坏肺的气体交换(SelmanM，KingTE，PardoA.Idiopathicpulmonaryfibrosis：prevailingandevolvinghypothesesaboutitspathogenesisandimplicationsfortherapy.AnnInternMed2001；134(2)：136-151；SimePJ，O′ReillyKM.Fibrosisofthelungandothertissues：newconceptsinpathogenesisandtreatment.ClinImmunol2001；99(3)：308-319.)，所以抑制成纤维细胞异常增殖很有意义。
中药续断具有补肝肾，强筋骨，续折伤，止崩漏的作用，用于腰膝酸软，风湿痹痛，崩漏，胎漏，跌扑损伤。酒续断多用于风湿痹痛，跌扑损伤，盐续断多用于腰膝酸软。续断皂苷X是从续断中提取获得的，为续断的有效成分，但续断皂苷X在制备预防或治疗肺纤维化的药理作用未见报道。本发明人通过大量的研究发现续断皂苷X可以通过抑制EMT和成纤维细胞异常活化从而在预防或治疗肺纤维化方面有明显的作用。基于此，本发明人发明了续断皂苷X通过抑制EMT和肺成纤维细胞异常活化从而抑制肺纤维化的发生发展，以达到预防或治疗IPF。
发明内容
本发明提供了续断皂苷X在制备预防或治疗肺纤维化的药物中的应用。
本发明提供了续断皂苷X通过抑制EMT达到预防或治疗肺纤维化的药物中的应用。
本发明提供了续断皂苷X通过抑制成纤维细胞异常活化达到预防或治疗肺纤维化的药物中的应用。
发明提供的药物在用于肺纤维化时，其使用剂量范围是50mg～2000mg；优选50～1000mg。
本发明还提供了由续断皂苷X和药学上可接受的载体或辅料组成的药物，该药物可以以药学常规方法制备成片剂、胶囊剂，优选以片剂形式存在。
具体实施方式
制备例1：制备川续断皂苷X
取川续断药材1公斤，将其粉碎，过10目筛，加10倍量(V/W)70％乙醇浸泡过夜，回流提取两次，每次2小时，提取液过滤，回收乙醇至无醇味，加水形成混悬液，用正丁醇萃取3次，合并下层水层，上样于处理好的D101大孔树脂(树脂与药材重量比1∶1)，水洗3个柱体积，弃去，再用30％乙醇洗脱3个柱体积，弃去，最后用50％乙醇洗3个柱体积，收集50％乙醇洗脱液，浓缩，活性炭脱色，重结晶，即得含量为98％的川续断皂苷X6.5g。
制备例2：制备川续断皂苷X
取川续断药材1公斤，将其粉碎，过10目筛，加10倍量(V/W)70％乙醇浸泡过夜，回流提取两次，每次2小时，提取液回收乙醇至无醇味，浓缩液加乙醇至醇浓度为80％，静置过夜；抽滤，滤液浓缩至无醇味，加5倍量水，静置过夜，抽滤，滤液上样于处理好的D101大孔树脂(树脂与药材重量比1∶1)，0.5％氢氧化钠溶液洗2个柱体积后用水洗至中性，继用30％乙醇洗脱3个柱体积，弃去，最后用50％乙醇洗3个柱体积，收集50％乙醇洗脱液，浓缩蒸干得粗提物；取上述粗提物上硅胶柱，以三氯甲烷—甲醇(3∶1)、(2∶1)、(1∶1)为溶剂进行梯度洗脱，收集三氯甲烷—甲醇(1∶1)洗脱液，浓缩，结晶，即得含量为98％的川续断皂苷X7.8g。
实施例3续断皂苷X片剂制备
称取50.0g续断皂苷X、55.0g糖粉、70.0g乳糖和23.0g羧甲基淀粉钠充分混合均匀后过100目筛，加入适量的3％PVPK30水溶液适量制软材，20目筛制粒，60℃干燥3小时，18目筛整粒，加入2.0g硬脂酸镁，混合均匀后浅凹冲压，调节片重约200mg，即得。
具体实施方式
试验例1续断皂苷X对TGF-β1刺激A549细胞钙粘蛋白表达的影响
1.1实验材料
DMEM培养基为Gibco公司产品，新生牛血清为杭州四季青公司产品，TGF-β1购自PeproTech公司，A549细胞购自中国典型培养物保藏中心，用含10％胎牛血清(Hyclone公司)的低糖DMEM培养基(GIBCO公司)，常规培养于5％的CO2，37℃人工培养箱中，细胞呈贴壁生长。E-cadherin一抗购买于sigma公司。
续断皂苷X(纯度98.5％，滨州医学院药物化学研究室制备)
1.2试验方法
当细胞生长融合至70％-80％，用无血清培养基饥饿24h，换新鲜培养液，将传代的A549细胞分为：(1)阴性对照组；(2)TGF-β1处理组(10μg/L作用时间48h)；(3)TGF-β1处理组(10μg/L作用时间48h)+续断皂苷X0.3μM；(4)TGF-β1处理组(10μg/L作用时间48h)+续断皂苷X1μM；(5)TGF-β1处理组(10μg/L作用时间48h)+续断皂苷X3Mm；(6)TGF-β1处理组(10μg/L作用时间48h)+续断皂苷X30μM。48h后收获并裂解细胞提取细胞总蛋白。BCA蛋白测定试剂盒(Pierce公司)测定蛋白浓度后，取50μg样品蛋白进行10％的十二烷基磺酸钠2聚丙烯酰胺(SDS-PAGE)凝胶变性电泳、然后转至聚偏二氟乙烯(PVDF)膜，加入上皮细胞钙粘蛋白(E-cadherin)一抗，β-actin作内参，Westernblot检测细胞E-cadherin(上皮细胞标记物)蛋白的表达。以TGF-β1未刺激组作为对照，分析E-cadherin蛋白的表达差异。
1.3试验结果
表1续断皂苷X对TGF-β1刺激A549细胞的E-cadherin表达影响(n＝6)
| 组别 | E-cadheri(％) |
| 正常对照 | 100±10.5 |
| TGF-β1刺激 | 30.8±5.2 |
| TGF-β1刺激+续断皂苷X 0.3μM | 42.5±8.9* |
| TGF-β1刺激+续断皂苷X 1μM | 48.5±7.5** |
| TGF-β1刺激+续断皂苷X 3μM | 50.5±7.5** |
| TGF-β1刺激+续断皂苷X 30μM | 56.8±10.1** |
*，P＜0.05，**，P＜0.01，与GF-β1刺激组比较
表1结果可以看出，TGF-β1刺激A549细胞48小时，上皮细胞标记物E-cad表达明显下降，说明TGF-β1刺激A549细胞可以在体外诱导肺泡上皮细胞向间质细胞转化，而加续断皂苷X0.3、1、3或30μM后48小时，明显抑制上皮细胞标记物E-cadherin表达的下降，说明续断皂苷X抑制肺泡上皮细胞向间质细胞转化。
试验例2续断皂苷X对TGF-β1刺激肺成纤维细胞株异常活化的影响
2.1实验材料
DMEM培养基为Gibco公司产品，新生牛血清为杭州四季青公司产品，TGF-β1购自PeproTech公司，肺成纤维细胞株购自中国典型培养物保藏中心，用含10％胎牛血清(Hyclone公司)的低糖DMEM培养基(GIBCO公司)，常规培养于5％的CO2，37℃人工培养箱中，细胞呈贴壁生长。
MTT检测试剂盒(上海碧云天生物技术有限公司)
续断皂苷X(纯度98.5％，滨州医学院药物化学研究室制备)
2.2试验方法
当细胞生长融合至80％-90％，用无血清培养基饥饿24h，换新鲜培养液，将传代的肺成纤维细胞分为：(1)阴性对照组；(2)TGF-β1处理组(10μg/L作用时间48h)；(3)TGF-β1处理组(10μg/L作用时间48h)+续断皂苷X0.3μM；(4)TGF-β1处理组(10μg/L作用时间48h)+续断皂苷X1μM；(5)TGF-β1处理组(10μg/L作用时间48h)+续断皂苷X3μM；(5)TGF-β1处理组(10μg/L作用时间48h)+续断皂苷X10μM；(6)TGF-β1处理组(10μg/L作用时间48h)+续断皂苷X30μM。48h后MTT检测试剂盒检测细胞增殖情况。以TGF-β1未刺激组作为对照，分析各组肺成纤维细胞增殖率的差异。
2.3试验结果
表2续断皂苷X对TGF-β1刺激肺成纤维细胞增殖影响(n＝6)
| 组别 | 细胞增殖(％) |
| 正常对照 | 100±15 |
| TGF-β1刺激 | 204±15 |
| TGF-β1刺激+续断皂苷X 0.3μM | 172±23* |
| TGF-β1刺激+续断皂苷X 1μM | 160±24* |
| TGF-β1刺激+续断皂苷X 3μM | 152±17** |
| TGF-β1刺激+续断皂苷X 30μM | 145±15** |
*，P＜0.05，**，P＜0.01，与TGF-β1刺激组比较
表2结果可以看出，TGF-β1刺激肺成纤维细胞48小时，肺成纤维细胞增殖率明显增加，说明TGF-β1刺激肺成纤维细胞可以在体外诱导肺成纤维细胞增殖，而加续断皂苷X0.3、1、3μM或30μM后48小时，明显抑制肺成纤维细胞增殖，说明续断皂苷X抑制肺成纤维细胞异常活化。
试验例3续断皂苷X对博莱霉素(BLM)诱导的肺纤维化模型大鼠的影响
3.1实验材料
博莱霉素购于上海拜力生物科技有限公司
羟脯氨酸检测试剂盒购自南京建成生物工程研究所
SD大鼠购买于滨州医学院实验动物研究中心
续断皂苷X(纯度98.9％，滨州医学院药物化学研究室制备)
3.2试验方法
60只大鼠，10％水合氯醛麻醉(350mg/kg，ip)，采用支气管内注射博莱霉素(5mg/kg)诱导肺纤维化模型。造模型2天后分组，分为模型组、续断皂苷X灌胃给药组1、续断皂苷X灌胃给药组2、续断皂苷X灌胃给药组3、续断皂苷X灌胃给药组4，连续给药4周。处死动物，取肺，称重，计算肺指数，形态学改变分级肺泡炎和肺纤维化分级按文献[SzapielSV，ElsonNA，FulmurJD，etal.Bleomycininducedinterstitialpulmonarydiseaseinthenude，athymicmouse[J].AmRevRespirDis，1979，120：893-899.]，将肺泡炎和肺纤维化的程度分为4级：0级无明显改变；1级轻度改变，病变范围小于全肺的20％；2级中度改变，病变范围占全肺的20％～50％；3级重度改变，病变范围占全肺的50％以上。测定肺组织羟脯氨酸含量。
3.3试验结果
表3续断皂苷X对博莱霉素(BLM)诱导的肺纤维化模型大鼠的影响
*，P＜0.05，**，P＜0.01，与模型组比较
表3结果显示续断皂苷X灌胃给药组1、续断皂苷X灌胃给药组2、续断皂苷X灌胃给药组3、续断皂苷X灌胃给药组4连续给药4周可以明显降低肺指数，降低纤维化分级，降低肺组织羟脯氨酸含量，说明续断皂苷X灌胃给药可抑制肺纤维化的发生发展。其中续断皂苷X灌胃给药组3(100mg/kg)与续断皂苷X灌胃给药组4(200mg/kg)比较，无明显差异。
Claims (1)
1.续断皂苷X在制备预防或治疗肺纤维化的药物中的应用，所述续断皂苷X在用于肺纤维化治疗时使用的剂量范围为100-1000mg。
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