CN104127543B - 一种用于治疗骨髓增生异常综合征的中药组合物及其应用 - Google Patents
一种用于治疗骨髓增生异常综合征的中药组合物及其应用 Download PDFInfo
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Abstract
本发明提供了一种用于治疗骨髓增生异常综合征的中药组合物及其应用。所述中药组合物质量组成如下:海金沙草10~50份,杜仲叶10~50份,白花蛇舌草40~90份。本发明的有益效果主要体现在:提供了一种能用来制备治疗骨髓增生异常综合征的新的药物目录,拓宽了病人的选药范围,为治疗骨髓增生异常综合征提供了新的候选药物。
Description
(一)技术领域
本发明涉及一种用于治疗骨髓增生异常综合征的中药组合物及其应用。
(二)背景技术
骨髓增生异常综合征(myelodysplasticsyndromes,MDS)是一组以骨髓无效造血伴外周血细胞一系、两系或多系减少为特征的恶性克隆性干细胞疾病,并具有向急性白血病转化的高风险,发生率高达3.3/100,000(CogleC.R.,CraigB.M.,RollisonD.E.,etal.Incidenceofthemyelodysplasticsyndromesusinganovelclaims-basedalgorithm:highnumberofuncapturedcasesbycancerregistries[J].Blood,2011,117(26):7121-5.)。目前新确诊的高风险MDS有上升趋势(SekeresM.A.,SchoonenW.M.,KantarjianH.,etal.CharacteristicsofUSpatientswithmyelodysplasticsyndromes:resultsofsixcross-sectionalphysiciansurveys[J].JNatlCancerInst,2008,100(21):1542-51.),唯一的治愈方案是异基因造血干细胞移植,但中老年患者不能耐受手术,只能接受小剂量的化疗,支持治疗和观察治疗(Szmigielska-KaplonA.,RobakT..Hypomethylatingagentsinthetreatmentofmyelodysplasticsyndromesandmyeloidleukemia[J].CurrCancerDrugTargets,2011,11(7):837-48.),但高危病人向白血病转化,其中位生存期只有0.4年(MalcovatiL.,GermingU.,KuendgenA.,etal.Time-dependentprognosticscoringsystemforpredictingsurvivalandleukemicevolutioninmyelodysplasticsyndromes[J].JClinOncol,2007,25(23):3503-10.)。1997年世界卫生组织推出MDS国际预后评估系统(IPSS),据骨髓的原始细胞比例、染色体核型和外周血三系减少程度将MDS分为低危组(0分),中危组(0.5-2分)和高危组(2.5分以上)。其中低危组治疗以细胞因子联合支持治疗,治疗重点在改善血液学异常,中危组和高危组常有多系病态造血和恶性克隆增生同时存在,治疗目标重点是减少向白血病转化的风险,改善预后,治疗手段主要为化疗联合细胞因子治疗,疗效差于急性髓系白血病(acutemyeloidleukemia,AML),异基因造血干细胞移植可有较好的效果,但移植相关的风险明显高于白血病(GreenbergP.L..Currenttherapeuticapproachesforpatientswithmyelodysplasticsyndromes.BrJHaematol,2010,150(2):131-43.EsteyE..Acutemyeloidleukemiaandmyelodysplasticsyndromesinolderpatients.JClinOncol,2007,25:1908-15.EsteyE.H.,ThallP.F.,CortesJ.E.,etal.Comparisonofidarubicin+ara-C-,fludarabine+ara-C-,andtopotecan+ara-C-basedregimensintreatmentofnewlydiagnosedacutemyeloidleukemia,refractoryanemiawithexcessblastsintransformation,orrefractoryanemiawithexcessblasts.Blood,2001,98(13):3575-83.)。但是由于大多数患者为老年人不能耐受骨髓移植,且移植相关风险较高,所以多数患者不得不依赖药物治疗和输血生存,并最终转化为AML而死亡,原发性AML是骨髓中异常的原始细胞(白血病细胞)大量增殖并浸润各种器官组织,使正常造血受抑制的血液系统的恶性肿瘤,常规化疗缓解率为55~72%,而高危MDS转白血病与原发性AML的生物学特性有很大差异,对化疗不敏感,易耐药,预后极差。近几年美国FDA批准的用于MDS治疗的去甲基化药物5-氮杂胞苷(5-aza-cytidine,AzaC)和5-氮杂-2’-脱氧胞苷(5-aza-2’-deoxycitidine,DAC),由于非靶向的细胞毒作用、潜在致癌性、临床应用后耐药病例的出现以及昂贵的治疗费用,限制了其在MDS的治疗应用(QinT.,CastoroR.,EIAhdabS.,etal.Mechanismsofresistancetodecitabineinthemyelodysplasticsyndrome.PLoSOne,2011,6(8):e23372)。因此寻找疗效佳、骨髓抑制轻且经济的新药来改善中高危MDS患者的预后,仍是目前研究MDS治疗的焦点。
(三)发明内容
本发明的目的是提供一种用于治疗骨髓增生异常综合征的中药组合物,并提供了具体的药效成分提取方法,成分明确,为新药筛选提供了候选药物。
本发明采用的技术方案是:
一种用于治疗骨髓增生异常综合征的中药组合物,其质量组成如下:
海金沙草10~50份
杜仲叶10~50份
白花蛇舌草40~90份。
所述的骨髓增生异常综合征为下列之一:难治性贫血(refractoryanemia,RA)、难治性贫血伴有环形铁粒幼细胞增多(refractoryanemiawithringsideroblasts,RARS)、难治性血细胞减少伴多系发育异常(refractorycytopeniawithmultilineagedysplasia,RCMD)、难治性贫血伴原始细胞增多Ⅰ型(refractoryanemiawithexcessblast-1RAEB-1)、难治性贫血伴原始细胞增多Ⅱ型(refractoryanemiawithexcessblast-1RAEB-2)、骨髓增生异常综合征不能分类(MDS-unclassified,MDS-U)、5q-综合征(MDSassociatedwithisolateddel(5q))、骨髓增生异常和骨髓增殖性疾病(MDS/myeloproliferativedisorder,MDS/MPD)。
本发明还涉及所述的中药组合物在制备治疗骨髓增生异常综合征的药物中的应用。
具体的,所述中药组合物中各中药原料经提取后用于制备药物,所述中药原料提取方法如下:
海金沙草提取:海金沙草干燥粗粉用70~80%乙醇加热回流提取2~3次,每次1~2h,合并提取液,减压浓缩得稠浸膏,加水成混悬液,依次用石油醚、氯仿、正丁醇萃取,取正丁醇萃取液,减压回收至干品,得正丁醇部位加粗硅胶拌样,上样于100~200目硅胶柱,以氯仿-甲醇梯度洗脱,所得流分减压回收至干,得海金沙草提取物;
杜仲叶提取:杜仲叶干燥粗粉,用70~80%乙醇加热回流提取2~3次,每次1~2h,合并提取液,减压浓缩得稠浸膏,加水成混悬液,依次用石油醚、乙酸乙酯、正丁醇萃取,取正丁醇萃取液,减压回收正丁醇至干品,干品加蒸馏水成混悬液,采用HPD450型大孔树脂进行动态吸附,然后用水洗脱至无色,再用5~10%乙醇洗脱,收集洗脱液,减压回收至干,得杜仲叶提取物;
白花蛇舌草提取:白花蛇舌草干燥粗粉,用80~90%乙醇加热回流提取2~3次,每次1~2h,合并提取液,减压浓缩得稠浸膏,加水成混悬液,依次用石油醚、乙酸乙酯、正丁醇萃取,取乙酸乙酯萃取液,减压回收乙酸乙酯至干品,干品加蒸馏水成混悬液,采用HPD300型大孔树脂进行动态吸附,然后用水洗脱至无色,再用60~70%乙醇以动态洗脱,收集,减压回收至干,得白花蛇舌草提取物;
将上述制得海金沙草提取物、杜仲叶提取物、白花蛇舌草提取物按照20~40:20~40:50~70质量比混合,即为所述治疗骨髓增生异常综合征的药物的药效成分。
所述药效成分每日有效剂量为6.25mg~100mg/kg成人。
所述药效成分添加本领域常规药用辅料,可制成下列之一的剂型:片剂、颗粒剂、丸剂、胶囊剂、口服液、散剂、注射剂。
本发明的有益效果主要体现在:提供了一种能用来制备治疗骨髓增生异常综合征的新的药物目录,拓宽了病人的选药范围,为治疗骨髓增生异常综合征提供了新的候选药物。
(四)附图说明
图1为PHDE对Skm-1细胞增殖的抑制作用;*表示组内比较有显著性差异P<0.05,#表示组间比较有显著性差异P<0.05。
图2-A为流式细胞术检测不同剂量PHDE诱导Skm-1细胞株凋亡图;图2-B为不同剂量PHDE对Skm-1细胞株凋亡率的影响,*P<0.05。
图3为经PHDE处理后Skm-1细胞凋亡形态学变化。
图4-A为不同剂量PHDE对Skm-1细胞周期影响图;图4-B为不同剂量PHDE对Skm-1细胞DNA含量(%)的影响,*P<0.05。
图5为不同剂量PHDE对Skm-1细胞凋亡相关蛋白表达的影响。
图6为不同剂量PHDE对Skm-1细胞IAPs相关蛋白表达的影响。
图7为不同剂量PHDE对Skm-1细胞Bcl-2家族相关蛋白表达的影响。
图8为不同剂量PHDE对Skm-1细胞周期相关蛋白表达的影响。
图9为不同剂量PHDE对Skm-1细胞信号通路相关蛋白表达的影响。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
实施例1:
海金沙草1Kg干燥粗粉,用12000mL的80%乙醇加热回流提取3次,每次2h,合并提取液,减压浓缩得100g稠浸膏。加500mL水成混悬液,依次用500mL石油醚、500mL氯仿、500mL正丁醇萃取三次。取正丁醇萃取液,减压回收至干品,得正丁醇部位(40g),加粗硅胶(800g)拌样,上样于硅胶(100~200目)柱,以氯仿-甲醇(80:20)洗脱,所得流分减压回收至干,得组分a11g。
杜仲叶1kg干燥粗粉,用10000mL的80%乙醇加热回流提取3次,每次2h,合并提取液,减压浓缩得稠浸膏130g。加500mL水成混悬液,依次用500mL石油醚、500mL乙酸乙酯、500mL正丁醇分别萃取三次。取正丁醇萃取液,减压回收正丁醇至干品,干品加100mL蒸馏水成混悬液,采用HPD450型大孔树脂纯化,按0.5mL/min的吸附速度(径高比为1:10)进行动态吸附,然后用水洗脱至无色,再用5%乙醇以1mL/min流速动态洗脱,收集洗脱液,减压回收至干,得组分b15g。
白花蛇舌草1Kg干燥粗粉,用12000mL80%乙醇加热回流提取3次,每次2h,合并提取液,减压浓缩得120g稠浸膏。加500mL水成混悬液,依次用500mL石油醚、500mL乙酸乙酯、500mL正丁醇各萃取三次。取乙酸乙酯萃取液,减压回收乙酸乙酯至干品,干品加100mL蒸馏水成混悬液,采用HPD300型大孔树脂,按0.5mL/min的吸附速度(径高比为1:10)进行动态吸附,然后用水洗脱至无色,再用60%乙醇以1mL/min流速动态洗脱,收集,减压回收至干,所得流分减压回收至干,得组分c10g。
上述组分a、组分b、组分c按照20:20:60质量比混合,得药效成分(PHDE)。
实施例2:中药组合物药效成分(PHDE)对骨髓增生异常综合征抑制的应用
(1)细胞培养
人MDS细胞株Skm-1细胞株由浙江大学医学院附属第一医院血液病研究所赠送,该细胞株从由日本学者Nakagawa从一名高危MDS病人建立的细胞株,细胞培养条件为含10%GIBCO胎牛血清的RPMI1640完全培养基,在37℃,5%CO2孵育箱中培养,每天传代一次。
(2)MTT法检测细胞存活率
取对数生长期的Skm-1细胞悬浮于RPMI1640培养基,计数后,按照5×104个/mL植入24孔培养板,每孔1mL。实验设加药组、对照组:加药组药物浓度分别为0.05、0.075、0.1、0.125mg/mL,对照组不加药,每个浓度设2个复孔。加过药物后将板置于37℃、5%CO2及饱和湿度的培养箱中培养,在指定的时间,转入96孔板中,每孔200μL,每个浓度4个复孔,每孔加入20μL5mg/mL的MTT工作液,37℃培养箱中继续孵育4h,离心(3000rpm,10min)后弃上清,每孔加入200μLDMSO溶液,吹打混匀后,于酶标仪570nm处读取吸光值(A),根据吸光值计算生长抑制率。结果见图1。
细胞增殖抑制率计算方法如下:细胞增殖抑制率=(1-A实验/A对照)×100%
利用MTT法,检测了PHDE作用24、48h对Skm-1细胞增殖的影响。结果如图1所示,作用24h后,0.05至0.125mg/ml的PHDE对Skm-1细胞的增殖抑制率分别为3.30%、15.83%、49.53%、67.93%,48h后各组细胞增殖抑制率为:4.70%、11.30%、54.30%、86.90%,由此表明,PHDE能显著抑制Skm-1细胞增殖,并且具有浓度依赖性。
(3)流式细胞术(FACS)检测细胞凋亡
取对数生长期的Skm-1细胞以1×105个/mL接种于六孔板,每孔体积为5mL,依次加入不同体积的PHDE,使每孔药物终浓度分别为0、0.075、0.1、0.125mg/mL,37℃、5%CO2培养箱中培养。24h后,1000rpm离心5min,弃去培养基,再用1mLPBS洗涤,共洗2次。然后每组各加入500μLAnnexinVBindingBuffer重悬细胞,再加入5μLAnnexinV-FITC和5μLPI,混匀,室温避光反应10min上机检测,采用FlowJo7.6软件分析结果。结果见图2-A。
为检测PHDE诱导Skm-1细胞凋亡情况,我们采用终浓度为0.075、0.1、0.125mg/mL的PHDE处理Skm-1细胞株,24h后收集细胞,利用流式细胞术(FACS)检测细胞凋亡。结果如图2-A所示,对照组早期凋亡率为4.49%,PHDE不同浓度处理后早期凋亡率为7.28%、9.37%、18.10%。结果表明,PHDE能有效诱导Skm-1细胞株凋亡。
(4)Hoechst33258染色观察细胞形态学变化
将对数生长期的Skm-1细胞离心后计数,以RPMI1640培养基稀释至1×105个/mL接种于六孔板,每孔体积为5mL,依次加入不同体积的PHDE,使每孔药物终浓度分别为0、0.075、0.1、0.125mg/mL,37℃培养箱中培养24h。离心(1000rpm,5min),收集细胞,PBS清洗2遍,每个处理组用500uL4%多聚甲醛冰上固定30min。PBS清洗2遍,将细胞涂至玻片,晾干,用2%Trixton-100溶液透化20min。倒掉多余液体,将玻片转移至摇床,PBS清洗3遍,每遍15min,将Hoechst33258溶液用PBS稀释1000倍后均匀滴加至玻片,室温避光孵育15min,PBS洗2遍,每遍15min,荧光显微镜下观察并拍照。结果见图3。
为了观察药物处理后Skm-1细胞形态学的变化,我们用0.05、0.075、0.1、0.125mg/mL的PHDE处理Skm-1后进行Hoechst33258荧光染色,并在荧光显微镜下观察、拍照。结果如图3,对照组细胞核形状规则,染色质分布均匀,细胞所发荧光较弱且均匀,而在药物处理组,细胞核出现皱缩、边集现象,可见致密强荧光,并且随着药物剂量增大,颗粒状或碎片状致密浓染逐渐增多。
(5)流式细胞术检测细胞周期阻滞
取对数生长期的Skm-1细胞离心(1000rpm,5min)后计数,用RPMI1640培养基稀释细胞密度至2×105个/mL接种于六孔板,每孔体积为5mL。加入PHDE使药物终浓度分别为0、0.05、0.075mg/mL,37℃培养箱中培养24h。每组收集1×106个细胞,1000rpm离心5min,弃去培液,PBS洗涤一次,再离心去除PBS,将细胞加入到预冷的70%乙醇中,4℃固定过夜。离心(1500rpm,5min)去除固定液,PBS重悬细胞,离心去除PBS。再用少量PBS重悬细胞,每组加入约2.5~5uL的RNaseA酶工作液使终浓度为50ug/mL,37℃温浴30min,再加入25~50uL的PI工作液使终浓度为50ug/mL,4℃避光反应30min后上机检测采用modfitLT软件分析,结果见图4-A。
通过流式细胞术PI染色法对细胞内DNA含量进行检测时,可以将细胞周期各时相区分为G1/G0,S期和G2/M期,并通过特殊软件,计算各时相的百分率。据此原理,我们分别用0.075、0.1mg/ml的PHDE处理Skm-1细胞,24h后收集细胞进行PI染色,FACS检测细胞周期。结果如图4-A所示,不加药物处理时,细胞G0/G1期比率为34.89%,S期为65.11%,0.075mg/ml的PHDE处理细胞后,G0/G1期细胞比率增至41.53%,S期细胞比率则下降至55.09%,0.1mg/ml的PHDE处理细胞后,G0/G1期细胞比率增至48.02%,S期细胞比率则下降至42.26%,提示PHDE可以使Skm-1细胞发生G0/G1期阻滞。
(6)Western-Blotting方法检测细胞凋亡相关蛋白、IAPs家族蛋白、Bcl-2家族相关蛋白、G0/G1期相关蛋白、PI3K/Akt信号通路蛋白及NF-κB信号蛋白
1).收集蛋白上清液
收集对数生长期的Skm-1细胞,用RPMI1640培养液稀释细胞密度至1×105个/mL接种于培养瓶中,每瓶体积为10mL。加入不同体积的PHDE使每个处理组药物终浓度分别为0、0.05、0.075、0.1mg/mL,37℃培养箱中培养24h。1000rpm离心5min收集细胞,根据细胞团块大小加入约30~40uL蛋白裂解液(含蛋白酶抑制剂),冰上放置30min后,4℃12000rpm离心10min,收集蛋白上清。
2).BCA蛋白定量法:取洁净的96孔板,在每个孔中加入1μL的蛋白标准品或蛋白样品,19μLPBS,每个孔中加入200μLBCA工作液,轻微震荡混匀30sec,60℃温育30min,取出96孔板,待冷却至室温后于酶标仪570nm波长检测吸光值,制作标准曲线,根据标准曲线计算蛋白样品浓度。
3).SDS-聚丙烯酰胺凝胶电泳
按Bio-Rad公司说明书安装玻璃板,分别灌制8%~12%分离胶和5%积层胶。凝胶固定于电泳装置中,上、下槽各加入1×Tris-甘氨酸电泳缓冲液,拔去梳子,并用电泳缓冲液冲洗梳孔,按30~50ug的蛋白量进行上样。浓缩胶用恒压60V,当样品跑至分离胶时,把电压调至120V,直至溴酚蓝跑至分离胶底部。根据胶的大小,剪四张合适的Whatman3mm滤纸和一张PVDF膜,PVDF膜先用甲醇浸泡活化,之后再和滤纸一起放在盛有转膜缓冲液的槽中浸泡5min,在黑色一面(负极)垫上海绵垫,在其上放置两张用转膜缓冲液浸泡过的滤纸,将凝胶置于其上,然后是PVDF膜和另外两张滤纸,将另一块海面垫置于其上,以上各步均应排尽气泡。将上述装置放入盛有转膜缓冲液的电转槽中,PVDF膜面朝正极,恒流400mA转100min左右。转移结束后将PVDF膜从电转槽中取出,浸泡在封闭液中,置摇床上缓慢摇动,室温2h。将PVDF膜转移到配制好的一抗溶液中,4℃过夜。用1×TBST洗膜三次,洗去非特异结合的蛋白,每次15min。PVDF膜正面朝上,加入现配的二抗溶液中,室温摇床孵育2h。用1×TBST洗膜三次,洗去非特异结合的蛋白,每次15min。根据膜的大小、多少,取等量的ECL发光试剂盒中的A液、B液进行混合,然后把PVDF膜置于混合液中淋洗1min。沥去膜上多余的溶液,将膜置于两层保鲜膜中,置于X光片盒中。在暗室中压片,曝光30s~5min,一次性完成显影、定影。
Caspase的激活是凋亡事件的核心机制,来自于细胞表面受体、线粒体和内质网的死亡诱导信号促发了Caspase级联反应,诱导细胞凋亡,Caspase-3是Caspase家族中最重要的执行分子,正常以酶原(32KD)形式存在于胞浆中,当接受Caspase启动分子的信号时被激活,裂解相应的胞浆核底物而导致细胞凋亡,多聚(ADP-核糖)聚合酶(poly(ADP-ribose)polymerase,PARP)是Caspase-3的主要底物,在DNA损伤修复、重组和维持基因组稳定性上起着重要的作用,在凋亡启动时,PARP被Caspase-3剪切成两个片段,使得PARP中与DNA结合的两个锌指结构与羧基端的催化区域分离,不能发挥正常功能而引起凋亡。我们用0.05~0.1mg/mLPHDE作用Skm-1细胞24h后,提取总蛋白,利用Western-Blotting检测了药物作用后Caspase家族相关蛋白的变化。结果显示如图5,PHDE能显著激活Caspase-8、Caspase-9、Caspase-3和PRAP等凋亡相关蛋白。
凋亡抑制蛋白(InhibitorofapoptosisproteinsIAPs)是一类抑制凋亡蛋白,其主要包括XIAP、CIAP-1、CIAP-2、Survivin、Livin等,是Caspases的主要调节因子之一。前面我们已经证实PHDE作用后引起Caspase家族蛋白的激活,是否IAPs也参与此调节过程,我们用0.05~0.1mg/mLPHDE作用Skm-1细胞24h后,提取总蛋白进行检测结果显示,PHDE显著抑制XIAP、CIAP-1、CIAP-2、Survivin等蛋白的表达(图6)。
我们观察到Caspase-9的激活,表明PHDE通过线粒体途径诱导细胞凋亡,在正常细胞中cytc位于线粒体内、外膜之间,在呼吸传递链中起重要作用,但当细胞被诱导凋亡是,cytc可以从线粒体释放进入细胞质,与Apaf-1结合,提高了Apaf-1结合ATP或dATP的亲和力,Apaf-1与cytc复合物与ATP/dATP的结合激发其多聚化从而形成凋亡体,在凋亡体上Apaf-1的CARD结构域向外暴露,吸引胱解酶9前体到凋亡小体,引起其他胱解酶的活化,进而导致凋亡。Bcl-2蛋白家族在线粒体通路中有重要的调节作用,Bcl-2是一个多基因家族,主要包括3个亚家族:Bcl-2亚家族(抑制细胞凋亡)主要包括Bcl-2、Bcl-xl、Mcl-1等,Bax亚家族(促进细胞凋亡)包括Bax、Bak,BH3-only蛋白家族,(促进细胞凋亡),包括Bid、Bad、Bim/Bod、Nip3、Nix/BNIP3等,我们用0.05~0.1mg/mLPHDE作用Skm-1细胞24h后,提取总蛋白进行检测结果显示,PHDE显著上调cytc,抑制Bcl-2亚家族(Bcl-2、Bcl-xl、Mcl-1)表达,并呈剂量依赖性,剂量依赖性上调Bax亚家族Bak,但对Bax、Bid表达无明显影响(图7)。
CyclinD/CDK4复合体是真核细胞必不可缺的G1/S期调控因素,是细胞周期推进的动力,CylcinD1是G1期活性最强的CyclinD亚型之一,被认为是细胞癌变的驱动器(GuglielmiF.,LuceriC.,GiovannelliL.,etal.Effectof4-coumaricand3,4-dihydroxybenzoicacidonoxidativeDNAdamageinratcolonicmucosa[J].BrJNutr,2003,89(5):581-7.)。CyclinD1和CDK4在多种肿瘤中均有高表达,因此,抑制CDK4,CyclinD1的表达使得细胞周期停滞在G1期可有效地抑制肿瘤生长,前期利用流式细胞术检测细胞周期发现PHDE诱导Skm-1细胞发生G1期细胞阻滞,因此我们检测调控细胞由G1期进入S期的CyclinD1、CDK4、CDK6表达。我们采用Western-Blotting检测了PHDE作用于Skm-1细胞24h后,CDK4/6、cyclinD1的变化。结果如图8所示,PHDE能显著下调CDK4/6、CyclinD1,并呈剂量依赖性。
PI3K/AKT信号通路为细胞内重要的信号转导通路之一,经线粒体通路抑制细胞凋亡促进细胞存活,PI3K、AKT是此通路中关键分子,NF-κB是一个转录因子蛋白家族,是与恶性肿瘤细胞转化、耐药和凋亡有关的重要因子。用0.05~0.1mg/mLPHDE作用Skm-1细胞24h后,提取总蛋白,Western-Blotting检测PI3K、Akt、p-Akt、p65和pp65蛋白的表达。结果显示,BIIB021显著抑制PI3K、磷酸化Akt(p-Akt)表达,对总Akt也有抑制作用,此外,PHDE也抑制p65和pp65蛋白表达(如图9)。
因此,本发明中药组合物药效成分(PHDE)能够抑制MDS细胞株Skm-1细胞增殖,且呈浓度-时间依赖性;能同时激活内、外源途径(死亡受体途径和线粒体途径)诱导Skm-1细胞凋亡;能将Skm-1细胞阻滞于G0/G1期,诱导细胞凋亡;能够通过PI3K/Akt信号通路,NF-κB信号通路而引起细胞凋亡,减少药物耐药性。
(7)统计学处理
数据用SPSS17.0软件分析,单变量两组间数据比较采取t-检验,所有实验均重复三次,各组数据以“均值±标准差”表示,以P<0.05为差异具有统计学意义。
Claims (5)
1.一种用于治疗骨髓增生异常综合征的中药组合物,其中药原料质量组成如下:
海金沙草10~50份
杜仲叶10~50份
白花蛇舌草40~90份。
2.如权利要求1所述的中药组合物在制备治疗骨髓增生异常综合征的药物中的应用。
3.如权利要求2所述的应用,其特征在于所述中药组合物中各中药原料经提取后用于制备药物,所述中药原料提取方法如下:
海金沙草提取:海金沙草干燥粗粉用70~80%乙醇加热回流提取2~3次,每次1~2h,合并提取液,减压浓缩得稠浸膏,加水成混悬液,依次用石油醚、氯仿、正丁醇萃取,取正丁醇萃取液减压回收正丁醇后,加粗硅胶拌样,上样于100~200目硅胶柱,以氯仿-甲醇梯度洗脱,所得流分减压回收至干,得海金沙草提取物;
杜仲叶提取:杜仲叶干燥粗粉,用70~80%乙醇加热回流提取2~3次,每次1~2h,合并提取液,减压浓缩得稠浸膏,加水成混悬液,依次用石油醚、乙酸乙酯、正丁醇萃取,取正丁醇萃取液减压回收正丁醇至干品,干品加蒸馏水成混悬液,采用HPD450型大孔树脂进行动态吸附,然后用水洗脱至无色,再用5~10%乙醇洗脱,收集洗脱液,减压回收至干,得杜仲叶提取物;
白花蛇舌草提取:白花蛇舌草干燥粗粉,用80~90%乙醇加热回流提取2~3次,每次1~2h,合并提取液,减压浓缩得稠浸膏,加水成混悬液,依次用石油醚、乙酸乙酯、正丁醇萃取,取乙酸乙酯萃取液减压回收乙酸乙酯至干品,干品加蒸馏水成混悬液,采用HPD300型大孔树脂进行动态吸附,然后用水洗脱至无色,再用60~70%乙醇以动态洗脱,收集,减压回收至干,得白花蛇舌草提取物;
将上述制得海金沙草提取物、杜仲叶提取物、白花蛇舌草提取物按照20~40:20~40:50~70质量比混合,即为所述治疗骨髓增生异常综合征的药物的药效成分。
4.如权利要求3所述的应用,其特征在于所述药效成分有效剂量为6.25mg~100mg/kg成人。
5.如权利要求3所述的应用,其特征在于所述药效成分添加药用辅料,制成下列之一的剂型:片剂、颗粒剂、丸剂、胶囊剂、口服液、散剂、注射剂。
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