CN115737634A - 鸦胆醇在制备抗nsclc药物中的应用 - Google Patents
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Abstract
本发明公开了一种鸦胆醇在制备抗癌药物中的应用，所述的抗癌药物以鸦胆醇为活性成分；所述抗癌药物用于治疗或者预防非小细胞肺癌。试验结果表明，鸦胆醇可以抑制NSCLC细胞的增殖，促进NSCLC细胞的凋亡，抑制NSCLC细胞迁移，因此，可以将鸦胆醇作为抗癌药物的活性成分，用于治疗或者预防非小细胞肺癌。
Description
技术领域
本发明属于药物制备领域，具体涉及一种鸦胆醇在制备抗NSCLC药物中的应用。
背景技术
根据世界卫生组织国际癌症研究机构(IARC)发布的2020年全球最新癌症负担数据显示，肺癌是全世界发病率第二、死亡率第一的恶性肿瘤；在我国，肺癌的发病率及病死率均位居第一，且肺癌死亡人数遥遥领先，高达71万，占癌症死亡总数的23.8％。非小细胞肺癌(Non-Small Cell Lung Cancer,NSCLC)是最常见的侵袭性肺癌，约占肺癌的85％，其中肺腺癌为NSCLC最常见亚型且所占的比例呈逐年增长的趋势。尽管近年来NSCLC的治疗取得重要突破，已由传统的手术治疗、化疗和放疗，发展到精准分子靶向治疗和免疫治疗，但由于大多数NSCLC患者在确诊时发生转移，其5年生存率仍低于16.1％，且近十几年无显著改善。
鸦胆子(Bruceajavanica)(图1A)为苦木科植物鸦胆子的干燥成熟果实，在传统中医药理论中，具有清热、解毒、截疟、止痢的作用，外用可腐蚀赘疣。鸦胆醇(Bruceantinol)是鸦胆子所含有的一种天然苦木内酯类化合物，现代药理学研究显示鸦胆醇具有抗炎、抗氧化、降血糖、增强免疫功能等多种药理活性(图1B)。近年来有研究证实鸦胆醇具有抗肿瘤活性。鸦胆醇可以发挥抗白血病的作用。在结肠癌中鸦胆醇与MEK抑制剂联合使用可阻断细胞抵抗的STAT3激活。鸦胆醇可通过内在线粒体凋亡途径诱导乳腺癌MCF-7细胞和MDA-MB-231细胞凋亡。到目前为止，鸦胆醇对NSCLC的抗肿瘤作用及其抗肿瘤机制尚未见报道。因此研究鸦胆醇的抗NSCLC的作用及其具体机制对于开发鸦胆醇的抗肿瘤药物有着重要的研究意义。
发明内容
本发明的目的在于提供一种鸦胆醇的新用途，为NSCLC的治疗提供新的药物治疗方案
本发明的技术方案如下：
一种鸦胆醇在制备抗癌药物中的应用，所述的抗癌药物以鸦胆醇为活性成分；
所述抗癌药物用于治疗或者预防非小细胞肺癌。
试验结果表明，鸦胆醇可以抑制NSCLC细胞的增殖，促进NSCLC细胞的凋亡，抑制NSCLC细胞迁移，因此，可以将鸦胆醇作为抗癌药物的活性成分，用于治疗或者预防非小细胞肺癌。
通过进一步的试验表明，鸦胆醇可能是通过激活早期反应因子2(Early growthresponse protein 2：EGR2)来达到治疗或者预防非小细胞肺癌的作用，其中，EGR2表达水平与NSCLC患者生存率正相关，鸦胆醇通过激活EGR2表达水平可提高NSCLC患者生存率。
EGR2是早期反应因子家族的一员，该家族是体内广泛存在的一类转录因子家族，参与多种基因的转录调控，与细胞的生长、增殖、分化以及凋亡等密切相关，是细胞信号转导传导的重要环节。EGR2蛋白质由406个氨基酸组成，含有3个Cys2His2锌指结构域，该结构域高度保守，入核后可与其他基因的启动子区域GCG G/T GGG CG结合从而调控下游基因的表达。既往研究发现EGR2对于后脑发育，外周神经髓鞘的形成的有积极作用，EGR2靶向某些特异性受体调节肿瘤微环境中功能失调的抗原特异性CD8+T细胞，增强机体免疫功能。且有研究证明EGR2在甲状腺癌、胃癌和乳腺癌等实体肿瘤中表达下降，且与恶性肿瘤的发生发展、转移情况、疾病预后及总生存期呈正相关。
附图说明
图1为鸦胆醇结构；
图2为实施例1中鸦胆醇抑制NSCLC细胞增殖的结果；
图3为实施例2中鸦胆醇促进NSCLC细胞凋亡的结果；
图4为实施例3中鸦胆醇抑制NSCLC细胞迁移；
图5为实施例4中鸦胆醇激活EGR2的结果；
图6为实施例4中NSCLC细胞中EGR2表达水平下降；
图7为实施例4中鸦胆醇促进EGR2及SOCS3表达。
具体实施方式
实施例1
本实施例通过EdU实验结果提示鸦胆醇能浓度依赖性抑制NSCLC细胞增殖(图2A)。进一步集落克隆实验结果显示鸦胆醇能抑制NSCLC细胞A549增殖(图2B)。
EdU实验：将3×104个/孔A549细胞接种在24孔板中，使其附着过夜，用DMSO(对照)、浓度梯度的鸦胆醇处理细胞12h。接下来按照EdU测定试剂盒说明方法处理细胞，并于倒置荧光显微镜下拍照。根据制造商的说明确定细胞增殖速率。使用以下公式计算EdU掺入率：率(％)＝(EdU掺入细胞数/Hechst 33342染色细胞数)×100％。
集落克隆实验：将1×103个/孔A549细胞接种在6孔板上并于37℃、5％CO2的培养箱中培养。两天后弃去原有培养基，用DMSO(对照)、浓度梯度的鸦胆醇处理细胞24h。后每两天替换新鲜培养基保持细胞生长10天。弃去培养基，菌落用PBS洗涤，用4％多聚甲醛在室温下固定15分钟后，再用PBS洗涤3次，用结晶紫染色10分钟。然后菌落再次用纯水洗涤，直到背景变成透明。当菌落干燥时，对菌落进行拍照。
实施例2
进一步利用Annexin V/PI双染法处理药物作用后的NSCLC细胞，再用流式细胞仪检测细胞凋亡的程度。发现鸦胆醇作用48h后，能诱导NSCLC细胞凋亡，结果呈剂量依赖性(图3A)。利用Hoechst 33324染料染色鸦胆醇处理24h后的PC-9细胞，同样验证以上结论(图3B)。同时，Western blot的结果显示鸦胆醇作用24h后，NSCLC细胞中促凋亡蛋白Bax、Cl-PARP1表达上调，抗凋亡蛋白Bcl-2表达下调(图3C)。
Annexin V/PI双染法：采用凋亡检测试剂盒(BD Biosciences,USA)检测细胞凋亡。HCC827细胞接种于6孔板。次日的贴壁细胞，用DMSO(对照)、浓度梯度的鸦胆醇处理48h，用预冷PBS洗涤两次后收集细胞，按照凋亡检测试剂盒的方法进行双染色，采用流式细胞仪检测细胞凋亡。
实施例3
我们采用了细胞划痕实验和Transwell迁移实验。结果所示，鸦胆醇能抑制NSCLC细胞的爬行能力，并呈现出浓度依赖性(图4A和4B)。
Transwell迁移实验：transwell小室是一个在24孔板(Corning，美国)中含有8.0um粒孔的聚对苯二甲酸乙二醇酯过滤器。将悬浮在200uL无血清培养基(3-4×104个细胞/mL)中的细胞移植到transwell小室的上室，下室填充含10％胎牛血清的培养基。在孵育过夜后，用DMSO(对照)、浓度梯度的鸦胆醇处理12h。随后，用棉签去除残留。4％多聚甲醛固定15min后,0.1％结晶紫染色20min。倒置显微镜观察拍照并计数侵袭细胞。
实施例4
为了进一步明确鸦胆醇抗NSCLC的具体作用机制，本实施例对NSCLC细胞系PC-9Control组及鸦胆醇加药处理组进行转录组测序分析，以Fold change>＝1.0和adj.P<0.05为阈值，共得到4194个差异基因，其中上调基因1649个，下调基因2545个。早期反应因子2(Early growth response protein 2：EGR2)在PC-9细胞鸦胆醇处理后显著表达上调，在所有差异基因中位居第一(图5)。
本实施例NSCLC细胞进行RT-qPCR及Western blot实验检测发现，发现和正常支气管上皮细胞BEAS-2B相比，NSCLC细胞中EGR2的表达水平下降(图6A、6B)。进一步利用Kaplan-Meier生存曲线分析TCGA数据库中1925个NSCLC患者肿瘤组织EGR2表达水平与生存率之间的关系，发现EGR2表达水平与NSCLC患者生存率正相关(图6C)。
计算机模拟对接提示鸦胆醇可以和EGR2蛋白的紧密结合(图7A)。进一步在NSCLC细胞系中加入浓度梯度的鸦胆醇，RT-qPCR和Western blot实验均提示鸦胆醇可浓度依赖性上调EGR2及其下游SOCS3的表达(图7B、7C、7D)。基于以上结果，我们推测鸦胆醇通过激活EGR2来发挥其抗NSCLC作用。
Claims (5)
1.一种鸦胆醇在制备抗癌药物中的应用，其特征在于，所述的抗癌药物以鸦胆醇为活性成分；
所述抗癌药物用于治疗或者预防非小细胞肺癌。
2.根据权利要求1所述的鸦胆醇在制备抗癌药物中的应用，其特征在于，所述的抗癌药物用于抑制NSCLC细胞的增殖。
3.根据权利要求1所述的鸦胆醇在制备抗癌药物中的应用，其特征在于，所述的抗癌药物用于促进NSCLC细胞的凋亡。
4.根据权利要求1所述的鸦胆醇在制备抗癌药物中的应用，其特征在于，所述的抗癌药物用于抑制NSCLC细胞迁移。
5.根据权利要求1所述的鸦胆醇在制备抗癌药物中的应用，其特征在于，所述的抗癌药物通过激活EGR2来达到治疗或者预防非小细胞肺癌的作用。
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