CN116617215A - 一种改善肺部疾病的组合物及其应用 - Google Patents
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Abstract
本发明公开了一种改善肺部疾病的组合物及其应用，属于生物医药技术领域。改善肺部疾病的组合物包括质量比为(1～2):(1～3)的L‑肌肽与麦角硫因，通过雾化给予COPD大鼠改善肺部疾病的组合物，验证了该组合物能够降低肺泡灌洗液中炎症因子TNF‑α、IL‑1β和IL‑6的水平，改善肺部炎症，提高大鼠肺部OCTN1的表达，抑制大鼠肺上皮细胞的凋亡，进而改善肺部损伤。
Description
技术领域
本发明属于生物医药技术领域，具体涉及一种改善肺部疾病的组合物及其应用。
背景技术
慢性阻塞性肺疾病(简称“慢阻肺”)是一种常见的慢性肺部疾病，一般包括慢性支气管炎和肺气肿。大多数患者由于长期接触有毒颗粒或气体(如香烟烟雾、空气污染等)从而导致肺损伤，肺损伤的主要症状为慢性咳嗽、咳痰、气短或呼吸困难等。
目前针对慢阻肺的常用西药主要有祛痰药或支气管扩张剂，可缓解气短、咳喘以及痰多的症状。但是该方法治标不治本，只能缓解症状，不能从根本上改善肺部炎症和抑制肺泡上皮细胞的凋亡，进而修复肺部损伤，因而需要一种能够有效治疗慢阻肺的药物。
发明内容
为了克服上述现有技术的缺点，本发明的目的在于提供一种改善肺部疾病的组合物及其应用，有效治疗慢阻肺。
为了达到上述目的，本发明采用以下技术方案予以实现：
本发明公开了一种改善肺部疾病的组合物，包括质量比为(1～2):(1～3)的L-肌肽与麦角硫因。
优选地，L-肌肽与麦角硫因的质量比为1:2。
优选地，还包括药用辅料或其它可配伍的药物。
本发明还公开了上述一种改善肺部疾病的组合物在制备改善肺部疾病药物中的应用。
优选地，所述肺部疾病为慢性支气管炎、支气管扩张症、肺气肿或肺纤维化。
优选地，所述药物的剂型为粉剂、片剂、丸剂、胶囊、口服液、喷雾剂、粉雾剂、气雾剂或滴鼻剂。
优选地，所述改善肺部疾病的组合物为降低肺泡灌洗液中炎症因子TNF-α、IL-1β和IL-6水平的组合物。
优选地，所述改善肺部疾病的组合物为提高肺部OCTN1的表达的组合物。
优选地，所述改善肺部疾病的组合物为抑制肺上皮细胞的凋亡的组合物。
优选地，L-肌肽与麦角硫因协同改善肺部疾病。
与现有技术相比，本发明具有以下有益效果：
本发明提供的一种改善肺部疾病的组合物，将麦角硫因和L-肌肽联用改善肺部损伤。该组合物能够1.有效降低肺部炎症和抑制肺上皮细胞凋亡；2.协同增效：L-肌肽减少肺泡灌洗液中炎症性细胞因子TNF-α、IL-1β、IL-6的水平，而炎性细胞因子是诱导或调节肺实质细胞凋亡的重要因素。麦角硫因主要通过肺细胞的转运蛋白OCTN1进入线粒体内，直接清除自由基，并保护线粒体，有效减少ROS诱导的炎症反应及减少线粒体介导的肺上皮细胞的凋亡。两者相互配合，协同增效，从不同途径抑制肺部炎症及肺上皮细胞的凋亡，极大增强了修复肺部损伤的能力；3.成分安全：食物中L-肌肽多来源于鸡肉、牛肉、猪肉中，麦角硫因来源于菌菇类以及麦角真菌，且人体内本身含有L-肌肽(肌肉和脑部的组织含有很高浓度的L-肌肽)和麦角硫因，故这两者本身都很安全。通过雾化给予COPD大鼠改善肺部疾病的组合物，进一步验证该组合物能够降低肺泡灌洗液中炎症因子TNF-α、IL-1β和IL-6的水平，改善肺部炎症，提高大鼠肺部OCTN1的表达，抑制大鼠肺上皮细胞的凋亡，进而改善肺部损伤。
附图说明
图1为本发明的COPD大鼠BALF中TNF-α、IL-1β、IL-6的水平图；其中，A为COPD大鼠BALF中TNF-α的水平，B为COPD大鼠BALF中IL-1β的水平，C为COPD大鼠BALF中IL-6的水平，a表示与Con组相比，p＜0.01；b表示与Mod组相比，p＜0.01；c表示与Car组和EGT组相比，p＜0.01；
图2为本发明的OCTN1的表达量的图；其中，A为通过ImageJ分析软件对蛋白表达条带定量，B为OCTN1蛋白表达条带，a表示与Con组相比，p＜0.01；b表示与Mod组相比，p＜0.01；c表示与Car组和EGT组相比，p＜0.01；
图3为本发明的肺上皮细胞的凋亡率的图；其中，a表示与Con组相比，p＜0.01；b表示与Mod组相比，p＜0.01；c表示与Car组和EGT组相比，p＜0.01。
具体实施方式
需要说明的是，本发明的说明书和权利要求书及上述附图中的术语“L-肌肽”，包括其别名“β-丙氨酰-L-组氨酸”“肌肽”等，均应该理解属于本发明保护的范围。
下面结合附图对本发明作进一步详细描述：
主要试剂及仪器：麦角硫因(纯度≥98％)购于深圳瑞德林生物技术有限公司；L-肌肽(纯度99％)、脂多糖(lipopolysaccharide，LPS)购于Sigma；大鼠TNF-α、IL-1β和IL-6ELISA试剂盒、TUNEL试剂盒，均购于武汉博士德公司；RIPA裂解液、BCA蛋白定量试剂盒、ECL显影液均购于上海碧云天；4％多聚甲醛购于北京鼎国；兔抗大鼠OCTN1抗体购于Biorbyt公司；兔抗大鼠β-actin抗体购于艾博抗(上海)贸易有限公司；山羊抗兔二抗购于北京中杉金桥生物技术有限公司；PARI JuniorBOY SX(085G3305)压缩雾化机购于德国百瑞公司；光学显微镜Olympus BX51购于Olympus公司。
动物及分组：36只体质量200±20g的SPF级SD雄性健康大鼠(由第四军医大学实验动物中心提供)，适应性饲养1周，温度22～26℃，湿度40％～50％，人工光照昼夜时间各12h，饲养期间饮水自由。将SD大鼠随机分为Con组(对照组)、Mod组(模型组)、Car组(L-肌肽组)、EGT组(麦角硫因组)、Mix组(L-肌肽:麦角硫因＝(1～2):(1～3)，优选1:2)，每组各6只。
实验方法：将Mod组、Car组、EGT组和Mix组大鼠于造模第1天、第14天时，用无菌注射器向气管内缓缓滴注LPS 200μg/200μL，将大鼠左右旋转，使LPS进入大鼠肺组织，对照组注入等体积的生理盐水；于2～13天、15～28天时，将Mod组、Car组、EGT组和Mix组大鼠置于密闭箱中进行烟熏，每天烟熏1次，每次12支烟，每次持续30min，得到COPD大鼠模型，且在烟熏前30min给药一次，Con组不作烟熏处理；给药方法为：分别将L-肌肽、麦角硫因、组合物(L-肌肽:麦角硫因＝1:2)溶于10mL生理盐水，给药剂量为80mg/kg，采用大鼠雾化给药仪分别对Car组、EGT组和Mix组大鼠进行雾化吸入治疗30min，Mod组及Con组吸入等体积的生理盐水。
肺泡灌洗液(BALF)中炎症因子的检测：于实验结束后次日，断颈处死大鼠后开胸，扎右侧主支气管，注入5mL无菌预冷PBS灌洗左侧支气管肺泡，共进行3次，灌洗液于4℃2500r/min离心15min，收集上清，随后置于-80℃中保存。用ELISA法检测BALF中TNF-α、IL-1β和IL-6细胞因子的水平，操作流程按照大鼠TNF-α、IL-1β和IL-6ELISA试剂盒说明书进行。
TUNEL法检测细胞凋亡：摘取大鼠肺组织，分离左肺组织并将其置于4％的多聚甲醛固定后进行蜡块包埋，将其制成厚度为4μm的石蜡切片，完成切片脱水后，按照TUNEL试剂盒检测说明书进行细胞凋亡检测。将切片封片后置于显微镜下观察，计算5个不重叠视野TUNEL阳性细胞比例的平均值，并计算细胞凋亡率。
肺组织OCTN1蛋白相对表达量的检测：RIPA裂解液研磨裂解大鼠右肺组织，提取肺组织总蛋白，随后利用BCA试剂盒测定其浓度，经SDS-PAGE电泳、膜转移至PVDF膜、封闭后，加入兔抗大鼠OCTN1抗体、兔抗大鼠β-actin抗体，于4℃条件下孵育过夜，TBS水洗3次，每次15min，加入经HRP标记的二抗(即山羊抗兔二抗)继续孵育1h，最后滴加ECL显影液对蛋白条带进行显影，观察各组蛋白条带成像情况，并通过ImageJ分析软件对蛋白条带进行分析。
结果分析参见图1～图3：
在香烟烟雾或其他刺激物刺激下，巨噬细胞和气道上皮细胞分泌TNF-α、IL-1β和IL-6等促炎细胞因子和趋化因子，诱导中性粒细胞和单核细胞的活化募集及其他细胞因子的释放，进而促进炎症；活化的中性粒细胞和巨噬细胞可通过释放氧自由基和蛋白质水解酶促进肺气肿的发展。气道炎性反应在慢阻肺疾病进展中具有重要意义，因此降低肺部炎症，可以有效改善肺部损伤。
图1中，相对于Con组，Mod组BALF中炎症因子TNF-α、IL-1β和IL-6的水平显著升高(p＜0.01)，表明大鼠COPD模型造模成功。与Mod组相比，Car组、EGT组、Mix组TNF-α、IL-1β和IL-6的水平均显著降低，说明L-肌肽、麦角硫因及两者的组合物均可以显著减低COPD大鼠肺部炎症。同时，Mix组降低炎症因子TNF-α、IL-1β和IL-6的效果明显好于Car组和EGT组，说明L-肌肽及麦角硫因在功效上协同增效，显著降低COPD大鼠的肺部炎症。
图2中，相对于Con组，Mod组肺组织OCTN1的表达显著降低(p＜0.01)。相反，与Mod组相比，Car组、EGT组、Mix组OCTN1的表达均显著升高，说明L-肌肽、麦角硫因及两者的组合物均可以显著提高COPD大鼠肺部OCTN1的表达。同时，Mix组提高OCTN1的效果明显好于Car组和EGT组，说明L-肌肽及麦角硫因在功效上协同增效，显著增加COPD大鼠肺部OCTN1的含量。
图3中，相对于Con组，Mod组肺组织肺上皮细胞凋亡率显著增加(p＜0.01)。相反，与Mod组相比，Car组、EGT组、Mix组肺上皮细胞凋亡率均显著下降，说明L-肌肽、麦角硫因及两者的组合物均可以抑制COPD大鼠肺上皮细胞的凋亡。同时，Mix组抑制肺上皮细胞凋亡的效果明显好于Car组、EGT组，说明L-肌肽及麦角硫因在功效上协同增效，显著抑制COPD大鼠肺上皮细胞的凋亡。
线粒体是机体内源性活性氧的主要产生部位，活性氧可以诱发炎症反应，同时线粒体还是内源性caspase-induced凋亡途径中的中心细胞器。OCTN-1是麦角硫因进入细胞和线粒体的转运蛋白，肺组织受损后，麦角硫因通过肺细胞的OCTN-1进入线粒体内，直接清除自由基，并保护线粒体，减少线粒体功能障碍诱发的炎症反应及抑制线粒体介导的肺上皮细胞的凋亡。相对于Mod组，EGT组、Mix组转运蛋白OCTN-1的表达显著升高，提示更多的麦角硫因可以通过转运体OCTN-1进入细胞和线粒体内，直接清除活性氧自由基，发挥抗氧化、保护线粒体DNA的作用，进而减少炎症和肺上皮细胞的凋亡。Car组由于减轻了肺部组织炎症，在一定程度上也减少了炎症造成的肺上皮细胞的凋亡。同时Mix组肺泡灌洗液中炎症因子及肺上皮细胞凋亡率明显低于Car组、EGT组，说明麦角硫因和L-肌肽可以协同增效，降低COPD模型大鼠肺泡灌洗液中炎症因子及肺上皮细胞凋亡率，且效果好于单一成分。
以上内容仅为说明本发明的技术思想，不能以此限定本发明的保护范围，凡是按照本发明提出的技术思想，在技术方案基础上所做的任何改动，均落入本发明权利要求书的保护范围之内。
Claims (10)
1.一种改善肺部疾病的组合物，其特征在于，包括质量比为(1～2):(1～3)的L-肌肽与麦角硫因。
2.根据权利要求1所述的一种改善肺部疾病的组合物，其特征在于，L-肌肽与麦角硫因的质量比为1:2。
3.根据权利要求1所述的一种改善肺部疾病的组合物，其特征在于，还包括药用辅料或其它可配伍的药物。
4.权利要求1～3任意一项所述的一种改善肺部疾病的组合物在制备改善肺部疾病药物中的应用。
5.根据权利要求4所述的应用，其特征在于，所述肺部疾病为慢性支气管炎、支气管扩张症、肺气肿或肺纤维化。
6.根据权利要求4所述的应用，其特征在于，所述药物的剂型为粉剂、片剂、丸剂、胶囊、口服液、喷雾剂、粉雾剂、气雾剂或滴鼻剂。
7.根据权利要求4所述的应用，其特征在于，所述改善肺部疾病的组合物为降低肺泡灌洗液中炎症因子TNF-α、IL-1β和IL-6水平的组合物。
8.根据权利要求4所述的应用，其特征在于，所述改善肺部疾病的组合物为提高肺部OCTN1的表达的组合物。
9.根据权利要求4所述的应用，其特征在于，所述改善肺部疾病的组合物为抑制肺上皮细胞的凋亡的组合物。
10.根据权利要求4所述的应用，其特征在于，L-肌肽与麦角硫因协同改善肺部疾病。
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