臭牡丹不同提取物的抗肿瘤活性筛选及其对裸鼠移植瘤中EMT相关蛋白的影响  

The Antineoplastic Activity Screening of Different Extracts from Clerodendrum bungei Steud and Their Effects on EMT-Related Proteins in Nude Mice with Transplanted Tumor

作　　者：余娜 唐林[2] 谢壮鑫 周厚元 唐皓[1] 李顺祥 朱克俭[1] Yu Na;Tang Lin;Xie Zhuangxin;Zhou Houyuan;Tang Hao;Li Shunxiang;Zhu Kejian(Hunan Academy of Chinese Medicine,Changsha 410006;Hunan University of Chinese Medicine,Changsha 410208)

机构地区：[1]湖南省中医药研究院,长沙410005 [2]湖南中医药大学第一附属医院,长沙410006 [3]湖南中医药大学,长沙410208

出　　处：《中药药理与临床》2020年第4期124-131,共8页Pharmacology and Clinics of Chinese Materia Medica

基　　金：中国博士后基金面上项目(编号:2019M652775);湖南省中医药管理局课题(编号:201935);中药学湖南省重点学科开放基(编号:zy201602);湖南省大学生创新项目(编号:306)。

摘　　要：目的:比较臭牡丹不同提取部位的抗肿瘤作用及其作用机制。方法:根据前期研究结果,采用乙酸乙酯、石油醚萃取、大孔树脂纯化方法,将臭牡丹苯乙醇苷类、黄酮类、萜类成分进行分离提取并纯化。建立CCK8、划痕试验、Transwell侵袭小室经典的抗肿瘤药理模型,检测臭牡丹三大活性部位对人肺腺癌A549体外增殖、迁移、侵袭的影响。并通过建立A549裸鼠移植瘤模型,检测各组裸鼠癌组织与癌旁组织中β-连环蛋白(β-catenin)、波形蛋白(Vimentin)、上皮细胞钙粘蛋白(E-cadherin)、糖原合成酶激酶-3(GSK-3β)、磷酸化糖原合成酶激酶-3蛋白(p-GSK-3β)蛋白的表达水平。结果:苯乙醇苷部位以类叶升麻苷和异类叶升麻苷为对照,峰面积归一化法测得苯乙醇含量为88.93%;黄酮类部位以芦丁为对照,紫外分光度法测得黄酮含量均值为60%;萜类以齐墩果酸为对照品,紫外分光光度法测得石油醚部位萜类成分含量为50.99%。CCK8法测得臭牡丹苯乙醇苷、黄酮、萜类成分的IC50平均值分别为0.125、0.46、1.43 mg/ml。划痕试验测得臭牡丹苯乙醇苷、黄酮、萜类成分的24 h愈合率分别为14.6%、16.9%、19.9%;48 h愈合率分别为22.4%、20.4%、29.7%,均明显降低(P<0.01)。与模型对照组相比,Transwell侵袭小室试验显示各药物干预组的穿膜细胞数均显著降低(P<0.01),0.4 mg/ml黄酮组与0.15 mg/ml苯乙醇苷组尤为显著。体内实验中,模型对照组瘤组织中的β-catenin、Vimentin蛋白表达增强(P<0.01),E-cadherin、GSK-3β、p-GSK-3β蛋白表达明显下调(P<0.05或P<0.01)。与模型对照组比较,臭牡丹0.2 g/kg组能下调瘤组织中的β-catenin、Vimentin蛋白表达(P<0.05),上调GSK-3β、p-GSK-3β(P<0.05)蛋白表达。结论:臭牡丹苯乙醇苷、黄酮、萜类成分均对人肺腺癌A549细胞的增殖、迁移与侵袭具有一定的抑制作用,其中苯乙醇苷部位、黄酮部位对A549细胞体外抑制作用均明�Objective: To investigate the antitumor effect of different extracts from Clerodendrum bungei Steud and their mechanism. Methods:According to the previous study results, the phenylethanol glycosides, flavonoids and terpenes of Clerodendrum bungei Steud were extracted by ethyl acetate, petroleum ether and purified by macroporous resin. Classical anti-tumor models like CCK8 assay,the scratch test and Transwell invasion models were established, the effects of three active parts from Clerodendrum bungei Steud on proliferation, migration and invasion of human lung adenocarcinoma A549 in vitro was detected. In addition, transplanted tumor in A549 nude mice model was established,the expression levels of βcatenin, Vimentin, Ecadherin, GSK3β, and pGSK3β in cancer tissues and adjacent tissues in the nude mice were detected.Results:In phenylethanol glycosides part, Glucosinolates and Heterosylglucoside as the reference compounds, the content of phenylethanol was 88.93%, which was measured by the area normalization method. In flavonoid parts, Rutin as the reference compound, the content of flavonoids was 60%, measured by ultraviolet spectrophotometry. In petroleum ether layer, Oleanolic acid as the reference compound, the content of terpenes was 50.99%, measured by ultraviolet spectrophotometry. CCK8 assay results showed that the IC 50 of phenylethanol glycosides parts, flavonoids parts and the terpenes parts were 0.125 mg/ml,0.46 mg/ml and 1.43 mg/ml respectively. The results in the scratch test showed that the 24 h healing rates in the 3 groups were 14.6%、16.9% and 19.9% respectively, the 48 h healing rates were 22.4%、20.4% and 29.7% respectively, all were significantly decreased(p<0.01). Compared with the control group, the results in the transwell invasion chamber test showed, the number of penetrating cells in each group was significantly decreased(P<0.01),which in 0.4 mg/ml flavonoids group and 0.15 mg/ml phenylethanol group showed the most significant difference. In vivo experiments, the protein expressions of

关 键 词：臭牡丹提取物 抗肿瘤 肺癌 上皮间质转化(EMT) Wnt经典型号通路 

分 类 号：R285.5[医药卫生—中药学]