CN113842391A - 三七皂苷r1的新用途 - Google Patents
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Abstract
本发明公开了三七皂苷R1的新用途，即其在制备治疗急性肺损伤药物中的应用，本发明通过腹腔注射脂多糖诱导小鼠急性肺损伤，建立急性肺损伤小鼠模型，研究了三七皂苷R1对小鼠急性肺损伤后肺组织病理改变以及相关指标的影响，实验结果证明了三七皂苷R1通过影响肺组织甲基CpG结合域2的表达，来缓解和治疗小鼠急性肺损伤，本发明为三七皂苷R1用于治疗急性肺损伤提供了初步依据。
Description
技术领域
本发明属于医药开发制备领域，特别涉及一种三七皂苷R1在制备急性肺损伤的药物中的应用。
背景技术
急性肺损伤（Acute lung injury, ALI）是各种直接和间接致伤因素，导致的肺毛细血管内皮细胞和肺泡上皮细胞损伤造成弥漫性肺间质及肺泡水肿，急性低氧性呼吸功能不全或呼吸衰竭，损伤到一定程度可以出现急性呼吸窘迫综合征（Acute respiratorydistress syndrome，ARDS）。ALI/ARDS 发病机制的实质是失控的炎症反应，由此导致弥漫性肺实质损伤和呼吸功能障碍是主要病理特征。肺损伤会引起活性氧内流增加导致炎症介质TNF-α、IL-6、IL-1β的释放，从而肺泡上皮细胞凋亡和基底膜剥脱，导致上皮细胞向间充质的转化增加，肺泡结构的改变，即异常的肺重塑导致肺泡内气体交换受损，随着病情进展导致间质性肺炎和肺纤维化。
近年来发现碱基的修饰能够稳定储存和传递遗传信息，这种以非序列改变所致基因表达变化为主要研究内容的学科称为表观遗传学，其中最具代表性的就是DNA甲基化。启动子区CpG岛甲基化可显著影响相关基因的表达，甲基CpG结合域蛋白选择性地与巨噬细胞中的Ship启动子结合，通过抑制Ship表达和增强PI3K/Akt信号可促进M2巨噬细胞的产生，进而影响肿瘤等恶性疾病的治疗。
三七皂苷R1来源于五加科人参属药用植物三七（Panax notoginseng），是三七中最具代表性的特征化合物之一，有抗凝血、抗炎、抗氧化和抗凋亡的作用。已有研究表明，三七皂苷 R1对急性心肌缺血和脂多糖诱导的心肌损伤的大鼠均具有保护作用。然而，关于三七皂苷R1治疗急性肺损伤的研究尚未报道。
发明内容
本发明提供了三七皂苷R1的新用途，本发明首先通过LPS诱导小鼠急性肺损伤模型，然后使用三七皂苷R1对急性肺损伤小鼠进行治疗，结果表明三七皂苷R1主要通过降低肺组织甲基CpG结合域2的表达来缓解小鼠急性肺损伤，本发明将三七皂苷R1应用在制备治疗急性肺损伤药物中，三七皂苷R1治疗急性肺损伤引起的肺泡结构破坏、氧化应激水平升高、肺组织炎性细胞侵润。
本发明治疗急性肺损伤是以三七皂苷R1为活性成分，用于制备治疗急性肺损伤药物，还可以加入一种或多种药物制剂上可接受的辅料，所述辅料包括药学领域常规的填充剂、稀释剂、粘合剂、赋形剂、吸收促进剂、填充剂、表面活性剂和稳定剂等，必要时还可加入香味剂、色素和甜味剂等；或者与其他活性成分复配发挥协同治疗急性肺损伤的作用；可以制成药剂学上适宜的使用剂型，例如胶囊、丸剂、粉剂、片剂、粒剂、口服液和注射液等多种形式。
三七皂苷R1经三七提取分离获得，其提取分离采用常规分离提取技术；本发明中使用的三七皂苷R1的纯度为90%。
在三七皂苷R1对急性肺损伤的治疗作用的药理活性研究中，通过动物实验对本发明加以证明，实验小鼠分为空白对照组、模型组、NG-R1的低、中、高剂量治疗组，实验采用腹腔注射LPS建立急性肺损伤小鼠模型，造模成功后，各治疗组每天灌胃给药连续三周后，处死所有实验小鼠，取肺组织，根据处死时小鼠体重和肺重计算其肺指数；检测肺组织中的MDA、SOD和GSH含量；肺组织经H&E染色后观察其组织病理学变化；实时荧光定量PCR检测NF- κB、IL-6、甲基CpG结合域2、SHIP、TGF-β1、α-SMA、COL-1的表达情况。结果表明，在肺部形态学上，相比正常组小鼠，模型组小鼠肺部严重肿大，组织病理切片结果也表明模型组小鼠肺泡结构严重破坏，呈现大量的炎性细胞侵润和纤维增生；与模型组相比，治疗组小鼠肺部肿大现象得到明显改善，肺泡结构逐渐趋于正常小鼠，肺部的胶原沉积和炎性坏死明显减少；同时，NG-R1高剂量治疗组小鼠肺部氧化应激水平较模型组小鼠显著下调；在基因水平上，治疗组小鼠肺部炎症因子IL-6表达显著下调；模型组中，甲基CpG结合域2的相对表达量明显高于正常小鼠，同时其下游因子SHIP等基因表达水平显著上升；在三七皂苷R1高剂量治疗组中，促纤维化因子α-SMA、COL-1表达水平接近正常小鼠。
以上研究结果表明，三七皂苷R1主要通过降低甲基CpG结合域2的表达来抑制氧化应激水平和炎症反应，最终发挥治疗小鼠急性肺损伤的作用。
附图说明
图1为三七皂苷R1对急性肺损伤小鼠肺指数的影响；与空白组比较，#P<0.05，##P<0.01；与模型组比较，*P<0.05，**P<0.01。
图2为小鼠组织病理学切片的H&E染色（400×）结果，其中A图为空白组，B图为模型组，C图为R1低剂量组，D图为R1中剂量组，E图为R1高剂量组。
图3为三七皂苷R1对急性肺损伤小鼠肺组织氧化应激水平的影响；#P<0.05，##P<0.01；与模型组比较，*P<0.05，**P<0.01，其中A图为MDA；B图为SOD，C图为GSH。
图4为三七皂苷R1对急性肺损伤小鼠炎症反应的影响；#P<0.05，##P<0.01；与模型组比较，*P<0.05，**P<0.01，其中A图为炎症相关因子NF-κB表达结果，B图为炎症相关因子IL-6表达结果。
图5为三七皂苷R1对急性肺损伤小鼠肺纤维化的影响；#P<0.05，##P<0.01；与模型组比较，*P<0.05，**P<0.01；其中图A为甲基CpG结合域2的表达结果，B图为SHIP的表达结果。
图6为三七皂苷R1对急性肺损伤小鼠肺纤维化的影响；#P<0.05，##P<0.01；与模型组比较，*P<0.05，**P<0.01，其中A图为TGF-β1的表达结果，B图为α-SMA的表达结果，C图为COL-1的表达结果。
具体实施方式
以下为本发明的具体实施方式，所述的实施例是为了进一步描述本发明，而不是限制本发明。实施例中的方法如无特殊说明均为常规方法，使用的试剂如无特殊说明均为常规市售试剂或按常规方法配置的试剂。
实施例1：动物实验的分组与给药
50只C57BL/6雄性小鼠，随机分为5组（每组10只）：空白对照组、模型组、三七皂苷R1治疗组包括低剂量组（50mg/kg）、中剂量组（100mg/kg）、高剂量组（200mg/kg）。模型组和治疗组小鼠一次性腹腔注射10mg/kg LPS，构建急性肺损伤小鼠模型；空白组小鼠一次性腹腔注射与模型组相同体积的生理盐水；造模后第二天，开始给药治疗，空白组和模型组每天灌胃蒸馏水，治疗组每天分别灌胃给予三七皂苷R1 50mg/kg、100mg/kg、200mg/kg，连续给药21天。
实施例2：样本收集
21天后处死所有实验小鼠，处死前称量小鼠体重，取出小鼠肺组织，生理盐水洗涤，擦去表面多余的水分，称重，观察小鼠肺部形态学变化，模型组小鼠较正常小鼠有严重的肺肿大现象；经三七皂苷R1治疗后，肺肿大的情况逐渐得到改善，根据处死时小鼠体重和肺重计算小鼠肺指数(mg/g)=肺重(mg)/小鼠体重(g)，结果如图1所示，模型组小鼠肺指数远高于各治疗组。
实施例3：小鼠肺组织病理学检测
肺组织经10%中性甲醛溶液固定后进行乙醇梯度脱水，二甲苯透明，石蜡包埋，切片（厚度4μm），切下的组织切片轻轻放在30%乙醇上进行第一次展片，然后用载玻片转移至45℃水浴锅中，第二次舒展，待切片完全展开时附于载玻片上；将切片立于支架上置于37℃烘箱烘干，用于后面的H&E染色。
H&E染色的具体操作步骤为：将烘干的切片分别放入二甲苯Ⅰ、二甲苯Ⅱ中，各处理5min，对切片上的石蜡进行溶解；从二甲苯中取出切片，按顺序移入无水乙醇、95%乙醇、80%乙醇、70%乙醇，各停留2min，最后流水清洗5min；将切片移入Harris苏木素溶液中浸泡5min，流水冲洗5min后，擦干切片上残余的染液；再在75%盐酸乙醇浸泡10s，流水冲洗片刻；将切片放置50℃水浴锅中保持5min，置于95%乙醇1min脱水，放入75%盐酸乙醇2min；按95%乙醇、无水乙醇的顺序进行脱水，各处理2min；将切片于二甲苯中透明5min；取出切片，晾干，在玻片中滴加树胶，然后用慑子加盖盖玻片，切片封好后，待其干燥后在光学显微镜下进行观察，结果如图2所示，正常小鼠肺泡结构清晰完整，而模型组肺泡结构严重破坏，呈现大量的炎性细胞侵润；三七皂苷R1高剂量组的肺泡结构基本完整，肺泡间隔稍有增厚，较模型组明显改善。
实施例4：小鼠肺组织氧化应激指标的检测
小鼠肺组织中的丙二醛（MDA）、超氧化物歧化酶（SOD）、谷胱甘肽（GSH）的含量检测所用试剂盒采购于南京建成生物工程研究所；从-80 ℃冰箱取出保存的小鼠肺组织样本，置于冰上化冻，然后用已灭菌处理的匀浆器将小鼠肺与生理盐水按质量比1:9的比例进行研磨，获得的匀浆液离心15min（4000rpm/min、4 ℃），取上清；然后按照试剂盒步骤说明分别检测肺组织中MDA、SOD、GSH的含量，结果如图3所示，MDA作为过氧化产物，在模型组小鼠中的水平明显上升，且远高于正常小鼠；在三七皂苷R1治疗组中MDA水平均有不同程度的下降，其中高剂量组最为显著（P<0.01）；SOD和GSH是两种重要的抗氧化酶，可以维持体内的氧化与抗氧化的平衡，但模型组中SOD和GSH的含量较正常组均大幅度降低，与模型组相比，三七皂苷R1组中二者的水平均有不同程度的回升。
实施例5：小鼠肺组织促炎因子表达水平的检测
使用购买于生工生物工程股份有限公司的动物全组织RNA提取试剂盒提取小鼠肺组织RNA，按照GoScriptTMReverse Transcriptase System逆转录合成cDNA第一链，反应体系和操作过程为：取5μg Total RNA，依次加入50ng oligo（dT）、2μL dNTP（2.5mM each）、DEPC水加至反应体积为14.5μL；混匀后，70℃加热变性5min后迅速在冰上冷却5min，然后依次加入4μL 5×First-stand buffer、0.5μLRNasin（200U）、1μL M-MLV（200U），混匀并短时离心，42℃温浴1.5h，取出后70℃加热10min，终止反应；以合成的第一链cDNA为模板进行RT-PCR分析，反应条件：95℃，15s；60℃，30s；72℃，30s；42个循环；每个样品3个重复，以β- actin作为内参基因，根据荧光定量PCR测量出的内参基因和目的基因的Ct值，采用实验组/对照组=2-ΔΔct为基因表达量计算TGF-β1、α-SMA、Col-1、IL-6和NF-κB的基因表达水平，其中ΔCt=实验组（目的基因Ct值–内参基因Ct值）–对照组（目的基因Ct值–内参基因Ct值）。使用SPSS软件进行分析。通过Primer Premier 5.0件设计引物如下：TGF-β1上游引物为：GTGCTAATGGTGGACCGCAACAA，下游引物为：TACGGGAAAGTGCCAGCCTCT；α-SMA上游引物为：TGACTGTGAGCATCCACTTCTG，下游引物为：GTGACTTCTTTTGTGATTGGGTTA；COL-1上游引物为：CGACTAAGTTGGAGGGAACGGCT，下游引物为：TGGCAGTTGCTAGGCACGAC；甲基CpG结合域2上游引物为：GGAGGAAGTGATCCGAAAATCAG,下游引物为：AGCATTTCCCAGGTATCTTGC；SHIP上游引物为：GAGACACTGTTTCAGCGTCTAC，下游引物为：CGTCTTCAAAAAGTCGGAATCCA；NF-κB上游引物为：GAGATTGACTTTTGTGCCCAGC，下游引物为：GAGAGAGCAGACAGACGGACGG；IL-6上游引物为：GAGGTGGAATCTCAGGGTTGTTTT，下游引物为：CAACTGAGGAATACTGAATGGCTGA；β-actin上游引物为：GATCATTGCTCCTCCTGAGC，下游引物为：ACATCTGCTGGAAGGTGGAC；
实时荧光定量PCR结果如下图4、图5、图6所示，其中图4为三七皂苷R1对急性肺损伤小鼠体内炎症因子的影响，可以看出模型组小鼠肺组织中炎症相关因子NF-κB、IL-6的表达上升，表明其炎症水平显著上升（P<0.01）；经三七皂苷R1治疗可以下调炎症因子的表达，其中高剂量组最显著（P<0.01）；由图5、6可以看出，三七皂苷R1治疗组中甲基CpG结合域2、SHIP等因子的表达均有下降趋势，且高剂量组下调水平显著（P<0.01）。三七皂苷R1通过影响甲基CpG结合域2的表达，进而调控SHIP、TGF-β1、α-SMA、COL-1与肺损伤相关因子，达到治疗急性肺损伤小鼠的作用。
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Claims (2)
1.三七皂苷R1在制备治疗急性肺损伤药物中的应用。
2.根据权利要求书1所述的应用，其特征在于：三七皂苷R1通过对肺组织甲基CpG结合域2表达的影响来实现应用。
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