CN116392466A - 一种乙酰紫草素在制备治疗肺纤维化药物中的应用 - Google Patents
一种乙酰紫草素在制备治疗肺纤维化药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种乙酰紫草素在药物制备中的应用，所述的药物用于预防或者治疗肺纤维化；所述的药物以乙酰紫草素为活性成分。其中，乙酰紫草素可以减轻肺组织的病理性改变、降低肺泡灌洗液中的总蛋白浓度、活细胞数/总细胞数比率和炎症因子的渗出、改善肺纤维化导致的肺功能障碍、降低肺部纤维化标志物的表达水平、降低细胞外基质沉积标志物和上皮间充质转化标志物的表达水平，降低肺泡上皮细胞的迁移能力，可以作为一种有效的肺纤维化治疗药物。
Description
技术领域
本发明属于药物制备领域，具体涉及一种乙酰紫草素在制备治疗肺纤维化药物中的应用。
背景技术
肺纤维化(pulmonary fibrosis)是以肺泡上皮细胞损伤、成纤维细胞增殖和大量细胞外基质(ECM)异常沉积为特征的间质性肺疾病，导致肺瘢痕形成和肺功能不可逆下降。肺纤维化发病原因有很多，包括环境因素、结缔组织疾病和某些药物的副作用等。绝大多数肺纤维化病人病因不明，被称为特发性肺纤维化(IPF)，特发性肺纤维化发病机制尚不清楚，在特发性间质性肺炎家族疾病中发病率最高，预后最差。
肺纤维化严重影响人体呼吸功能，表现为干咳、进行性呼吸困难，且随着病情和肺部损伤的加重，患者呼吸功能不断恶化。特发性肺纤维化发病率和死亡率逐年增加，死亡率高于大多数肿瘤，被称为一种“类肿瘤疾病”。目前，IPF尚无有效治疗方法，现有的治疗肺纤维化的药物吡非尼酮(PFD)和尼达尼布都并不能从根本上治疗肺纤维化，且伴有腹泻等的胃肠道和皮肤疾病的副作用。因此，深入探讨肺纤维化的发病机制，寻找其他有效的药物已成为临床急需解决的问题。
乙酰紫草素(图1)是一种萘醌衍生物，主要从紫草中分离得到在中国传统医学中，紫草被广泛使用了1000年，主要来自中国的新疆。紫草的活性成分具有多种药理活性，目前，乙酰紫草素的药理活性如抗癌、抗炎、抗氧化、抗菌、抗真菌、调节脂质、抗糖尿病、神经保护和抗病毒等。此外，之前的研究表明，紫草对不同细胞的炎症具有有效的抑制作用，并抑制炎症小体和NF-κB的激活。此前也有研究证明乙酰紫草素通过抑制TGF-β1/Smad途径改善糖尿病小鼠的肾纤维化。对于乙酰紫草素的研究大多在它的抗肿瘤活性上，乙酰紫草素对肺纤维化的作用尚不清楚。因此研究乙酰紫草素的抗肺纤维化活性有助于我们找到新的治疗肺纤维化的药物。
发明内容
本发明提供了一种乙酰紫草素在制备治疗肺纤维化药物中的应用，为肺纤维化的治疗和预防提供了新的药物选择。
本发明的技术方案如下：
一种乙酰紫草素在药物制备中的应用，所述的药物用于预防或者治疗肺纤维化；
所述的药物以乙酰紫草素为活性成分。
试验结果表明，乙酰紫草素可以减少减轻肺组织的病理性改变和肺组织的胶原沉积；可以降低肺部水肿程度、肺部胶原组织沉积和纤维化程度；可以降低肺泡灌洗液中总蛋白浓度、活细胞数/总细胞数比率、降低炎症因子IL-1β、IL-6、TNF-α、CXCL1/IL-8的渗出；改善肺功能障碍，降低弹性阻力和呼吸阻力，提高肺顺应性。
进一步的试验表明，乙酰紫草素可以降低纤维化标志物(α-SMA)的表达水平，降低TGF-β1刺激后Beas-2b细胞中细胞外基质沉积标志物Collagen I、Fibronectin、α-SMA和上皮间充质转化(EMT)标志物Vimentin、N-Cadherin和基质金属蛋白酶(MMP-2)的表达水平，降低TGF-β1刺激后Beas-2b细胞的迁移能力，降低TGF-β1刺激后A549和MLE-12细胞迁移能力。
上述结果表明，乙酰紫草素可以作为一种有效的治疗肺纤维化的潜在药物。
附图说明
图1为乙酰紫草素的化学结构式和它的天然来源紫草；
图2为乙酰紫草素对肺纤维化小鼠体重和病死率的影响；
图3为实施例1中乙酰紫草素对肺纤维化小鼠肺组织病理性改变的影响；
图4为实施例2中乙酰紫草素对肺纤维化小鼠肺部水肿程度和胶原蛋白含量的影响；
图5为实施例3中乙酰紫草素对肺纤维化小鼠肺泡灌洗液各项指标的影响；
图6为实施例4中乙酰紫草素对肺纤维化小鼠肺功能的影响；
图7为实施例5中乙酰紫草素对肺纤维化小鼠肺部纤维化标志物的影响；
图8为实施例6中乙酰紫草素对Beas-2b细胞活力及TGF-β1刺激后细胞外基质沉积标志物和EMT标志物的影响；
图9为实施例7中乙酰紫草素对Beas-2b细胞迁移能力的影响；
图10为实施例8中乙酰紫草素对A549和MLE-12细胞在TGF-β1刺激后细胞外基质沉积标志物和EMT标志物的影响；
图11为实施例8中乙酰紫草素对A549和MLE-12细胞迁移能力的影响。
具体实施方式
小鼠肺纤维化模型的建立及指标测定
本发明采用单次气管滴注博来霉素(3mg/Kg)建立小鼠肺纤维化模型，造模后一天进行腹腔注射剂量为1mg/kg和2mg/kg的乙酰紫草素治疗，每两天注射一次，并于造模前及每次给药前记录小鼠体重。造模28天后处死，进行肺组织收集以及各项指标的测定，结果表明乙酰紫草素可以减轻博来霉素诱导的小鼠肺纤维化。
实施例1
按照前文的方法建立肺纤维化模型，于造模前及每次给药前记录小鼠体重，结果见图2，图2中，BLM表示仅注射博来霉素不注射乙酰紫草素组，CON为正常对照组。通过体重曲线可以发现乙酰紫草素对小鼠体重没有影响(图2A)；通过生存曲线发现乙酰紫草素可以减轻肺纤维化小鼠的病死率(图2B)。
实施例2
按照前文的方法建立肺纤维化模型，进行肺组织收集，取右肺中间叶置于4％多聚甲醛中固定48小时后进行石蜡包埋、切片、脱蜡、水化、苏木素-伊红染色、脱水、封片等步骤后，通过HE染色发现乙酰紫草素可以减轻肺组织的病理性改变(图3A)。同样取右肺中间叶置于4％多聚甲醛中固定48小时后进行石蜡包埋、切片、脱蜡、水化、重铬酸钾铬化处理、丽春红染色、磷钼酸处理、苯胺蓝染色、1％冰醋酸分化、脱水、封片等步骤后，通过Masson染色发现乙酰紫草素可以减少肺组织的胶原沉积(图3B)。
实施例3
按照前文的方法建立肺纤维化模型，28天处死，收集肺泡灌洗液后取出肺组织，小心剪去心脏和周围其他脂肪组织，将肺置于预冷的PBS中漂洗血液，吸干残余液体后，取右肺上叶和左肺下叶置于空锡纸盒中称量肺湿重后，于60℃烘箱放置48小时后称量肺干重，计算肺湿/干重比，通过肺湿/干重比发现乙酰紫草素可以降低肺纤维化小鼠肺部水肿程度(图4A)；取肺组织30-100mg，采用碱性水解法测定肺组织羟脯氨酸含量，操作步骤按试剂盒说明书进行，通过肺组织羟脯氨酸含量测定发现乙酰紫草素可以降低肺纤维化小鼠肺部胶原组织沉积和纤维化程度(图4B)。
实施例4
按照前文的方法建立肺纤维化模型，收集各组小鼠肺泡灌洗液(BALF)，4℃，3000rpm，离心10min，吸取上清液测定BALF中总蛋白浓度，我们发现乙酰紫草素可以降低肺纤维化小鼠BALF中总蛋白浓度(图5A)；之后将沉淀重悬于50uL PBS中，用细胞计数板进行细胞计数，我们发现乙酰紫草素可以降低肺纤维化小鼠BALF中活细胞数/总细胞数比率(图5B)。我们将BALF上清液做ELISA检测，发现乙酰紫草素可以降低肺纤维化小鼠BALF中炎症因子IL-1β(图5C)、IL-6(图5D)、TNF-α(图5E)、CXCL1/IL-8(图5F)的渗出。以上实验证明了乙酰紫草素可以改善小鼠肺纤维化过程中伴随的炎症表现。
实施例5
按照前文的方法建立肺纤维化模型，在造模后28天对各组小鼠进行了肺功能各项指标的测定，结果发现乙酰紫草素可以改善博来霉素引起的小鼠肺功能障碍，降低弹性阻力(图6A)和呼吸阻力(图6B)，提高肺顺应性(图6C)。
实施例6
按照前文的方法建立肺纤维化模型，然后对小鼠肺组织进行切片进行免疫组化实验，结果发现乙酰紫草素可以降低纤维化标志物(α-SMA)的表达水平(图7A)。以上实验均证明了乙酰紫草素可以减轻博来霉素诱导的小鼠肺纤维化。
实施例7
为检测药物对细胞活力的影响，取生长状态良好、同时处于在对数生长期的人支气管上皮(Beas-2b)细胞，对细胞进行消化，并用完全培养基对细胞进行重悬，配成3×104个/ml细胞悬液。设置5个复孔，每孔取100μl铺于96孔板中，周围加一圈PBS，在37℃、5％CO2恒温培养箱中培养24小时。显微镜下观察贴壁后细胞密度，等到细胞密度达到60-80％时，更换200μL的新鲜培养基。以DMSO为溶剂配制所需浓度梯度的药物溶液(1、2、4、5、8、10、20、50μM)，于96孔板内加药干预(设置阴性对照组只加入DMSO)，置于37℃、5％CO2恒温培养箱中培养24小时。药物干预结束后，取出96孔板，吸去各孔上清液，用PBS洗2次。避光条件下，配置CCK8反应液(DMEM基础培养基:CCK8反应液＝10:1)，每孔加入110μl CCK8反应液。加入反应液后，用锡纸包裹96孔板于培养箱中孵育1小时。孵育结束后，用酶标仪在450nm波长下测定OD值。本实施例通过CCK8实验筛选出不影响细胞活力的药物浓度(图8A)，最终将2μM作为Beas-2b后续细胞实验的作用浓度。
通过WB实验发现乙酰紫草素可以降低TGF-β1刺激后Beas-2b细胞中细胞外基质沉积标志物Collagen I、Fibronectin、α-SMA和上皮间充质转化(EMT)标志物Vimentin、N-Cadherin和基质金属蛋白酶(MMP-2)的表达水平。(图8B)。
实施例8
Transwell实验取对数生长期的Beas-2b细胞，对细胞进行消化，加入完全培养基重悬细胞；Corning 8.0μm 24孔板小室放入24孔板中，将100μl3×104个/ml细胞悬液加入Transwell小室上室，下室加入600μl基础培养基，贴壁培养12-18小时，分组为正常组(CON)、单加药物组(2μM AS)、造模组(10ng/ml TGF-β1)、治疗组(10ng/ml TGF-β1+2μMAS)，下排孔加入600μl对应的反应液，将小室移入反应液中，上室补充100μl基础培养基；置入培养箱中培养24h；造模结束后，吸出小室内的液体，放入4％多聚甲醛中固定30min；将小室取出，风干后置于结晶紫中染色30min，棉签去除多余的结晶紫；使用普通光学显微镜100倍视野进行观察和拍照，使用Image J软件对图像进行定量统计分析。
划痕实验先用marker笔在6孔板背后均匀地划横线，大约每隔1-1.5cm一道，横穿过孔，每孔穿过3条线；在孔中加入1ml 5×105个/ml细胞悬液，分组与干预的方式如前所述，待细胞长满后，用200μl枪头尽量垂直于背后的横线划痕，每孔划3条线；用PBS洗3次，去除划下的细胞，加入新鲜的完全培养基，分组与干预的方式如前所述；放入37℃、5％CO2恒温培养箱中培养24小时、48小时；使用普通光学显微镜100倍视野在0、24、48小时进行观察和拍照，使用Image J软件对图像进行定量统计分析。通过transwell(图9A)和划痕实验(图9B)证明乙酰紫草素可以降低TGF-β1刺激后Beas-2b细胞的迁移能力。
实施例9
我们后续又用人肺泡上皮(A549)和小鼠肺泡上皮细胞(MLE-12)进行了如前所述的WB实验、划痕实验和transwell实验，A549细胞分组与Beas-2b细胞的分组一样，MLE-12细胞分组为正常组(CON)、单加药物组(10μM AS)、造模组(10ng/ml TGF-β1)、治疗组(10ng/mlTGF-β1+10μM AS)，为验证乙酰紫草素对A549和MLE-12细胞TGF-β1刺激后细胞外基质沉积标志物和EMT标志物的影响(图10A、B)，验证了乙酰紫草素对A549和MLE-12细胞迁移能力的影响(图11A、B)。得出与上述一样的结果。所以基于上述实验结果，可以认为乙酰紫草素是一种抑制肺纤维化的药物。
Claims (8)
1.一种乙酰紫草素在药物制备中的应用，其特征在于，所述的药物用于预防或者治疗肺纤维化；
所述的药物以乙酰紫草素为活性成分。
2.根据权利要求1所述的乙酰紫草素在药物制备中的应用，其特征在于，所述药物用于减轻肺组织的病理性改变。
3.根据权利要求2所述的乙酰紫草素在药物制备中的应用，其特征在于，所述药物用于减轻肺纤维化的病死率。
4.根据权利要求1所述的乙酰紫草素在药物制备中的应用，其特征在于，所述药物用于降低肺泡灌洗液中的总蛋白浓度、活细胞数/总细胞数比率和炎症因子的渗出。
5.根据权利要求1所述的乙酰紫草素在药物制备中的应用，其特征在于，所述药物用于改善肺纤维化导致的肺功能障碍，包括：降低弹性阻力和呼吸阻力，提高肺顺应性。
6.根据权利要求1所述的乙酰紫草素在药物制备中的应用，其特征在于，所述药物用于降低肺部纤维化标志物Collagen I、Fibronectin和α-SMA的表达水平。
7.根据权利要求1所述的乙酰紫草素在药物制备中的应用，其特征在于，所述药物用于降低上皮间充质转化标志物和基质金属蛋白酶的表达水平。
8.根据权利要求1所述的乙酰紫草素在药物制备中的应用，其特征在于，所述药物用于降低肺泡上皮细胞的迁移。
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