CN115105537A - 金骨莲制剂在制备治疗或预防肺损伤药物中的应用 - Google Patents
金骨莲制剂在制备治疗或预防肺损伤药物中的应用 Download PDFInfo
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Abstract
本发明属于药物应用技术领域，具体涉及金骨莲制剂在制备治疗或预防肺损伤药物中的应用，所述金骨莲制剂由透骨香、汉桃叶、大血腾、八角枫、金铁锁制备而成，所述制剂为胶囊剂、颗粒剂、片剂、合剂、丸剂、喷雾剂等剂型。研究表明金骨莲制剂具有显著降低脂多糖致急性肺损伤大鼠血清中的TNF‑α、IL‑1β、IL‑6及PGE2表达水平，降低肺湿干重比，降低NF‑κBp65、P‑IκBα/IκBα蛋白表达水平，减轻急性感染引起的肺部水肿，可治疗或预防肺损伤。
Description
技术领域
本发明涉及药物应用技术领域，尤其是金骨莲制剂在制备治疗或预防肺损伤药物中的应用。
背景技术
肺损伤是由不同致伤因素导致的肺实质的损伤，肺部外伤、肺部手术、肺部感染、肺栓塞等都可导致肺损伤。肺损伤多是由于肺部感染、肺部手术愈合不佳引起。肺损伤的主要症状是胸痛、咯血、气促、呼吸困难、气胸、低热。
急性肺损伤(acutelunginjury，ALI)是各种因素导致的肺泡上皮细胞及毛细血管内皮细胞损伤，造成弥漫性肺间质及肺泡水肿，导致的急性低氧性呼吸功能不全，以肺容积减少、肺顺应性降低、通气/血流比例失调为病理生理特征。临床多种疾病均可引起急性肺损伤，其病因包括上呼吸道感染、肺炎、严重脓毒症、胃食管反流、大量输血、淹溺、休克、脂肪和羊水栓塞胰腺炎、肺挫伤、水杨酸盐/麻醉药过量、肺泡出血、烟雾和有毒气体吸入、再灌注损伤等疾病。
目前，国内外公认治疗ALI的方案：(1)去除病因；(2)在积极治疗原发病的基础上，尽早机械通气来纠正缺氧和改善组织供氧； (3)药物治疗；临床上广泛使用肾上腺糖皮质激素、糖皮质激素、环磷酰胺、甲胺蝶呤等治疗急性肺损伤，能减少毛细血管渗出和抑制肺纤维化的形成，使肺功能得到快速的改善；但糖皮质激素等免疫抑制剂对急性肺损伤选择性差，长期应用会产生多种并发症和副作用，严重影响患者生活质量，不适宜长期使用， 因此，寻找选择性高、毒副作用小的治疗肺损伤药物具有重要的临床意义。
金骨莲制剂，由透骨香、汉桃叶、大血腾、八角枫、金铁锁制备而成，可制成胶囊剂、颗粒剂、片剂、合剂、丸剂、喷雾剂、涂膜剂、软膏剂、贴剂等剂型。具有祛风除湿,消肿止痛。用于风湿痹阻所致的关节肿痛、屈仲不利等，金骨莲胶囊目前是已上市销售多年的治疗风湿骨痛的常用药。但在治疗或预防肺损伤的作用还未见报道。
发明内容
本发明的目的在于提供金骨莲制剂在制备治疗或预防肺损伤药物中的新用途。
本发明所述的肺损伤为感染性炎症引起的肺损伤。所述肺损伤包括上呼吸道感染、慢性支气管炎、肺气肿、慢性阻塞性肺疾病、哮喘、支气管扩张症、呼吸衰竭、肺炎、肺脓肿、肺结核、心脑缺血以及器官移植引起的肺组织损伤、炎症和感染。其主要症状为：肺组织水肿、肺泡间隔病变、炎症细胞浸润。
本发明所述金骨莲制剂，按重量份计，制成该制剂的有效组分为：透骨香170-500份、汉桃叶200-600份、大血藤180-550份、八角枫25-75份、金铁锁25-75份。
优选地，所述金骨莲制剂的有效组分为：透骨香343克、汉桃叶400克、大血 藤370克、八角枫50克、金铁锁50克。
本发明所述金骨莲制剂，是按一定工艺制备而成，其制备方法属于现有技术，可以从现有专利公开的文件中获得，或根据国家药品标准制备。如CN 03117137.0、WS-10093(ZD-0093)-2002-2012Z等。
优选为：取金铁锁加水煎煮3小时，滤过，滤液备用；药渣干燥后粉碎成细粉，备用；再将透骨香、汉桃叶、大血藤、八角枫四味，加水煎煮三次，每次2小时，合并煎液，滤过，合并上述金铁锁滤液，浓缩成清膏;加入一种或多种药学上可接受的辅料，制备成所需要的剂型。
所述金骨莲制剂加入的可接受辅料为稀释剂、助溶剂、缓释剂、吸收促进剂、崩解剂、湿润剂、粘合剂、表面活性剂，按照药学领域的常规方法，制备成所需要的剂型。
所述金骨莲制剂的剂型为胶囊剂、颗粒剂、片剂、合剂、丸剂、喷雾剂等剂型。优选为胶囊剂。
本发明所述的金骨莲制剂在使用时根据病人的情况确定用法用量。如已上市的由贵州益佰制药股份有限公司的金骨莲胶囊，规格：每粒装0.25g，用法用量: 口服。 一次 2粒，一日3次；或遵医嘱。
为了探究金骨莲制剂治疗肺损伤的用途，本发明进行了药效学研究，比较给药后大鼠血清中的TNF-α、IL-1β、IL-6及PGE2表达水平、肺湿干重比等指标变化。经动物实验验证，结果显示本发明所述的金骨莲制剂具有显著降低脂多糖致急性肺损伤大鼠血清中的TNF-α、IL-1β、IL-6及PGE2表达水平，降低肺湿干重比，降低NF-κBp65、P-IκBα/IκBα蛋白表达水平，表明其具有治疗或预防肺损伤的作用。
附图说明：
1.图1为各组大鼠肺组织观察结果（HE染色，×400）
2.图2为各组对大鼠血清TNF-α分泌的影响(ng/L，±SD，n = 8)
3.图3为各组对大鼠血清IL-1β分泌的影响(ng/L，±SD，n = 8)
4.图4为各组对大鼠血清IL-6分泌的影响(ng/L，±SD，n = 8)
5.图5为各组对大鼠血清PGE2分泌的影响(ng/L，±SD，n = 8)
6.图6为各组对大鼠肺组织湿干比的影响(mg/g，±SD，n = 8)
7.图7为各组肺组织TNF-α的mRNA表达水平（±SD，n = 3）
8.图8为各组肺组织IL-6的mRNA表达水平（±SD，n = 3）
9.图9为各组肺组织GE2的mRNA表达水平（±SD，n = 3）
10.图10为各组肺组织IL-1β的mRNA表达水平（±SD，n = 3）
11.图11为各组肺组织NF-κBp65的蛋白表达水平（±SD，n = 3）
12.图12为各组肺组织P-IκBα/IκBα的蛋白表达水平（±SD，n = 3）
13.图13为各组肺组织NF-κBp65、P-IκBα/IκBα的蛋白表达水平(±SD,n = 3)
14.图1中A为空白对照组，B为模型对照组，C为地塞米松组，D为金骨莲低剂量组，E为金骨莲中剂量组，F为金骨莲高剂量组
15.图2--图13中Con：空白对照组， Mod：模型对照组，J-L：金骨莲低剂量组，J-M：金骨莲中剂量组，J-H：金骨莲高剂量组，Dex：地塞米松组
16.与模型对照组比较，*P＜0.05，**P＜0.01，***P＜0.001。
具体实施方式
下面结合具体的实施方式来对本发明的技术方案做进一步的限定，但要求保护的范围不仅局限于所作的描述。
实验例一：金骨莲胶囊对脂多糖诱导的大鼠急性肺损伤保护作用研究
一、试验材料
1、 药品与试剂：
金骨莲胶囊（清膏）；LPS（大肠杆菌血清型O55：B5，Sigma公司）；醋酸地塞米松（规格：0.75 mg，批号：171007）；TNF-α、IL-6、IL-1β以及PGE2试剂盒（南京建成生物技术有限公司，批号：20191111）；Trizol（TAKARA，批号：9109）；iScript cDNA Synthesis Kit（Bio-Rad，批号：1708890）；iTaqTM universal SYBR Green Supermix（Bio-Rad，批号：1725124）；PierceTM Rapid Gold BCA Protein Assay Kit（Elabscience，批号：E-BC-K318-M）；5×蛋白上样缓冲液（Siwega，批号：8190011128）；TRIS（BioFROxx，批号：115KG001-1KG）；甘氨酸（BioFROxx，批号：1275KG2P5-2.5KG）；SDS（Solarbio，批号：#S8010-500g）；ECL化学发光底物（Bio-Rad，批号：170-5060）。
2、 动物 健康SD雄性大鼠，体重为（300±20）g，由长沙天勤生物技术有限公司提供（SCXK(湘)2014-0011），实验室级别SPF级。
二、试验方法：
1 造模、分组与给药
将48只雄性SD大鼠随机分为6组（n =8）,分别为空白对照组、模型对照组、金骨莲胶囊低、中、高剂量组（0.66 g×kg-1 、1.32 g×kg-1 、2.64 g×kg-1）、地塞米松组（0.945mg×kg-1）。给药组大鼠灌胃给与相应药物，空白对照组和模型对照组对应给与等体积空白溶媒，每12 h给药1次，预先连续给药3天。于末次给药后30 min除空白对照组外各组腹腔注射LPS（10 mg×kg-1），空白对照组腹腔注射等体积生理盐水，腹腔注射，6 h后出模。
测定细胞因子
尾静脉取血，室温自然凝固20分钟，3000 rpm离心20 min，分离血清，-20℃分装保存。用酶联免疫法（ELISA ）根据制造商的方法测定各组大鼠血清中TNF-α、IL-1β、 IL-6以及PGE2的含量。
肺湿重/干重(W/D)测定
取大鼠右肺中下叶，滤纸吸干表面水分后称重，为湿重（W）。然后将肺组织放置于80℃恒温干燥箱烘烤48 h后称重，为干重（D）。计算肺组织湿重干重(W/D)比值以反映肺组织的水肿程度。
肺病理学检测
取下大鼠左肺，以10 %甲醛固定，常规脱水、石蜡包埋、切片后，苏木精-伊红（HE）染色，在光学显微镜下观察大鼠肺泡间隔病变、炎症细胞浸润等肺组织病理学变化。
检测肺组织TNF-α、IL-1β、IL-6、及PGE2的表达
将大鼠肺组织RNA提取后，逆转为eDNA进行 RT-PCR检测，引物由北京擎科生物科技有限公司合成。引物如下：TNF-α引物：forward 5-'GGGGGCCACCACGCTCTTCTGTC-3'，reverse 5-' GCTGCTCCTCCGCTTGG TGGTTT -3'，IL-1β引物：forward 5-'CAGCTTTCGACAGTGAGG AGA-3'，reverse 5-'TTGTCGAGATGCTGCTGTGA-3'，IL-6引物：forward 5-'GTTTCTCTCCGCAA GAGACTTC-3'，reverse 5-'TGTGGGTGGTATCCTCTGT GA-3'，PGE2引物：forward 5-'CCCTGCCTTTCACAATCTTTGC-3'，reverse 5-'CCGACAACAGAGG ACTGAGC-3'，反应程序：95℃变性15s，60℃退伙15s，72℃延伸15s，循环数45个。3次独立重复实验。
检测肺组织NF-κBp65、IκBα、p-IκBα蛋白表达
利用蛋白质印迹法评估NF-κBp65、IκBα、p-IκBα蛋白表达水平。各组大鼠肺组织部分剪碎后加入蛋白提取试剂，提取蛋白质，利用组织匀浆仪制备组织匀浆，放冰上裂解30min，离心取上清为蛋白样，蛋白跑胶上样，经过电泳转膜封闭后，将封闭后的PVDF膜装入杂交袋中4°C孵育过夜，TBST洗脱后敷二抗。滴加新鲜配制的ECL混合溶液到膜的蛋白面侧，发光检测，用AlphaEaseFC软件处理系统分析目标带的光密度值。
统计学处理
采用SPSS 18.0软件进行统计学分析，所有数据均以±SD表示，采用单因素方差分析进行多组间样本均数比较，组间的两两比较采用LSD检验，P <0.05表示差异有统计学意义。
三、试验结果
1、肺病理学检测结果
选用HE染色方法评价肺组织的形态学改变。图1A为空白对照组大鼠肺组织病理切片, 肺泡结构清晰，肺泡壁薄，肺间质无炎症细胞浸润；图1B为模型对照组肺组织病理切片，肺间质和肺泡内水肿伴不等量的红细胞渗出和纤维素沉积，大量炎症细胞浸润，肺泡间隔增厚，成纤维细胞增生；图1C、图1D、图1E、图1F、分别为地塞米松组、金骨莲低、中、高剂量组肺组织病理切片，病理显示肺泡壁毛细血管均有轻度扩张，肺泡腔大小、形态、数量及分布未见异常，局部伴少量炎细胞浸润，间质血管轻度扩张充血，局部肺泡间隔轻度增厚，四组肺组织病理切片病变程度不同减轻。
、ELISA测定细胞因子结果
空白对照组大鼠进行腹腔注射生理盐水后，精神状态良好，行动自如，进食正常；其余各组大鼠注射LPS后出现精神不振、反应迟钝、腹泻等现象。取各组大鼠血清应用ELISA法测定TNF-α、IL-1β、IL-6以及PGE2水平，模型对照组与空白对照组比较，模型对照组大鼠血清中TNF-α、IL-1β、IL-6以及PGE2的含量均升高（P<0.01），提示LPS提高了大鼠血清中炎症细胞因子的表达，表明模型制备成功。与模型对照组比较，金骨莲中剂量组和高剂量组及地塞米松组大鼠血清中TNF-α、 IL-1β、 IL-6以及PGE2的含量均显著性降低（P<0.05），提示金骨莲中剂量组、高剂量组及地塞米松组可以有效地降低由LPS所致的炎性细胞因子的表达。结果见图2—图5。
、肺湿重/干重(W/D)测定结果
模型对照组与空白对照组比较，模型对照组大鼠肺组织W/D比值显著性升高（P <0.01），提示模型对照组大鼠肺组织出现了明显的水肿现象。金骨莲各剂量组、地塞米松组与模型对照组比较，金骨莲低、中剂量组W/D比值降低，但不具有统计学意义。金骨莲高剂量组及地塞米松组的肺组织W/D比值显著性降低（P <0.05），提示金骨莲胶囊高剂量组及地塞米松能够有效地降低由LPS所致的肺组织水肿。结果见图6。
、 RT-PCR检测肺组织TNF-α、IL-6、PGE2及IL-1β的表达结果
模型对照组与空白对照组比较，模型对照组大鼠肺组织TNF-α、IL-6、PGE2及IL-1βmRNA均显著性升高（P <0.05），提示模型对照组大鼠肺组织出现了明显的炎症反应。金骨莲各剂量组、地塞米松组与模型对照组比较，金骨莲中剂量组和地塞米松组TNF-α、IL-6、PGE2及IL-1β mRNA表达水平均降低（P <0.05）。提示金金骨莲中剂量组及地塞米松组能够有效地降低由LPS所致的肺组织炎症反应。结果见图7-图10。
、 WB检测肺组织NF-κBp65、IκBα、p-IκBα蛋白表达结果
模型组大鼠肺组织NF-κBp65、P-IκBα/IκBα蛋白水平明显高于空白对照组（P <0.01）；金骨莲各剂量组、地塞米松组与模型对照组比较，金骨莲中剂量组、高剂量组和地塞米松组大鼠肺组织NF-κBp65、P-IκBα/IκBα明显低于模型组（P <0.05）。结果见图11-图13。
四、结论：
本研究发现金骨莲胶囊可抑制大鼠肺组织中TNF-α、IL-1β、IL-6、及PGE2的表达，可以减轻LPS诱导的大鼠炎症反应，具有较好的抗感染性炎症作用，表明金骨莲胶囊能减轻肺损伤，治疗或预防肺损伤。其作用机制可能与抑制NF-κB通路有关。
实施例二：金骨莲胶囊的制备
称取金铁锁500克，加6倍量水煎煮3小时，滤过，滤液备用；药渣干燥后粉碎成细粉，备用；再称取透骨香3430克、汉桃叶4000克、大血藤3700克、八角枫500克，加6倍量水煎煮3次，每次2小时，合并煎液，滤过，合并上述金铁锁滤液，浓缩成相对密度1.25-1.28(60℃)的清膏；加入淀粉适量，混匀，干燥，粉碎，过筛，装入胶囊，制成10000粒。
实施例三：金骨莲片的制备
称取金铁锁500克，加5倍量水煎煮3小时，滤过，滤液备用；药渣干燥后粉碎成细粉，备用；再称取透骨香3430克、汉桃叶4000克、大血藤3700克、八角枫500克，加4倍量水煎煮3次，每次2小时，合并煎液，滤过，合并上述金铁锁滤液，浓缩成相对密度1.25-1.28(60℃)的清膏；加入淀粉适量，混匀，制粒，干燥、过筛，压片，制成10000片。
在此有必要指出的是，以上实验例和实施例仅限于对本发明的技术方案做进一步的阐述和理解，不能理解为对本发明的技术方案做进一步的限定，本领域技术人员作出的非突出实质性特征和显著进步的发明创造，仍然属于本发明的保护范畴。
Claims (10)
1.金骨莲制剂在制备治疗或预防肺损伤药物中的应用。
2.如权利要求1所述的应用，其特征在于，所述肺损伤为感染性炎症引起的肺损伤。
3.如权利要求1所述的应用，其特征在于，所述肺损伤包括上呼吸道感染、慢性支气管炎、肺气肿、慢性阻塞性肺疾病、哮喘、支气管扩张症、呼吸衰竭、肺炎、肺脓肿、肺结核、心脑缺血以及器官移植引起的肺组织损伤、炎症和感染。
4.如权利要求1所述的应用，其特征在于，所述肺损伤主要症状为：肺组织水肿、肺泡间隔病变、炎症细胞浸润。
5.如权利要求1所述的应用，其特征在于，所述的金骨莲制剂由如下重量份比例的原料药制成：透骨香170-500份、汉桃叶200-600份、大血藤180-550份、八角枫25-75份、金铁锁25-75份。
6.如权利要求1所述的应用，其特征在于，所述的金骨莲制剂由如下重量份比例的原料药制成：透骨香343份、汉桃叶400份、大血藤370份、八角枫50份、金铁锁50份。
7.如权利要求1-6 任一项所述的应用，其特征在于，所述金骨莲制剂是根据需要加入药学上可接受的辅料，所述辅料为稀释剂、助溶剂、缓释剂、吸收促进剂、崩解剂、湿润剂、乳化剂、粘合剂、表面活性剂中的一种或多种，按照药学领域的常规方法，制备成所需要的剂型。
8.如权利要求7所述的应用，其特征在于，所述金骨莲制剂的剂型为胶囊剂、颗粒剂、片剂、合剂、丸剂、喷雾剂等剂型。
9.如权利要求8所述的应用，其特征在于，所述金骨莲制剂的剂型为胶囊剂。
10.如权利要求9所述的应用，其特征在于，所述金骨莲制剂的制备方法为：取金铁锁加水煎煮3小时，滤过，滤液备用；药渣干燥后粉碎成细粉，备用；再将透骨香、汉桃叶、大血藤、八角枫四味,加水煎煮3次，每次2小时，合并煎液，滤过，合并上述金铁锁滤液，浓缩成清膏；加入金铁锁细粉及适量淀粉，混匀，干燥，粉碎，过筛，装入胶囊，即得。
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| CN105193835A (zh) * | 2015-10-26 | 2015-12-30 | 天津市南开医院 | 鹅掌楸苷及其苷元在制备防治脓毒症所致肺损伤药物中的用途 |
| CN112107609A (zh) * | 2020-11-10 | 2020-12-22 | 贵州益佰制药股份有限公司 | 金骨莲制剂在制备治疗睑板腺囊肿药物中的应用 |
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| CN105193835A (zh) * | 2015-10-26 | 2015-12-30 | 天津市南开医院 | 鹅掌楸苷及其苷元在制备防治脓毒症所致肺损伤药物中的用途 |
| CN112107609A (zh) * | 2020-11-10 | 2020-12-22 | 贵州益佰制药股份有限公司 | 金骨莲制剂在制备治疗睑板腺囊肿药物中的应用 |
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