芪玉三龙汤调节Beclin1,Atg5,LC3B表达以抑制肺癌肿瘤生长机制  

Effect of Qiyu Sanlong Decoction in Inhibiting Growth of Lung Cancer by Regulating Expressions of Beclin1,Atg5 and LC3B

作　　者：程建超[1] 童佳兵 朱洁[1,2] 张星星 高雅婷 陈杨[1] 李泽庚 郑莉莉[2] CHENG Jian-chao;TONG Jia-bing;ZHU Jie;ZHANG Xing-xing;GAO Ya-ting;CHEN Yang;LI Ze-geng;ZHENG Li-li(Key Laboratory of Traditional Chinese Medicine(TCM)Prevention and Treatment of Pulmonary Diseases of Anhui Provincial Education Department,Anhui University of Chinese Medicine,Hefei 230038,China;Research Institute of Respiratory Diseases of TCM,Anhui Academy of TCM,Hefei 230031,China)

机构地区：[1]安徽中医药大学,安徽省教育厅中医药防治肺系重大疾病重点实验室,合肥230038 [2]安徽省中医药科学院中医呼吸病防治研究所,合肥230031

出　　处：《中国实验方剂学杂志》2020年第9期29-35,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(81874431);国家自然科学基金青年基金项目(81804039);安徽省自然科学基金项目(1808085QH256);安徽中医药大学科研项目(2018zrzd19)。

摘　　要：目的:研究芪玉三龙汤对肺癌小鼠皮下移植瘤生长及对自噬关键分子酵母Atg6同系物(Beclin1),自噬相关基因5(autophagy related genes,Atg5)及微管相关蛋白1轻链3(microtubule-associated protein1light chain3,LC3B)表达的影响。方法:采用Lewis肺癌细胞(lewis lung cancer cell,LLC)构建肺癌移植瘤小鼠模型,造模成功后随机分为模型组、芪玉三龙汤组、顺铂组及联合组,每组18只移植瘤小鼠。模型组每日按照生理盐水20 mL·kg^-1灌胃;芪玉三龙汤组每日按照80.48 g·kg^-1灌胃;顺铂组在第1,3,5天腹腔注射顺铂溶液(DDP)0.4 mL;联合组每日按80.48 g·kg^-1灌胃给药,并分别在第1,3,5天腹腔注射顺铂溶液0.4 mL;连续治疗21 d剥离瘤组织,称取瘤重,计算抑瘤率。苏木素-伊红(HE)染色观察肿瘤组织形态学改变。免疫组化检测肿瘤组织中Beclin1,LC3B蛋白表达和定位。蛋白免疫印迹法(Western blot)测定Beclin1,Atg5,微管相关蛋白1轻链3-I(LC3B-Ⅰ)及微管相关蛋白1轻链3-Ⅱ(LC3B-Ⅱ)蛋白表达,并计算LC3B-Ⅱ/LC3B-Ⅰ。实时荧光定量聚合酶链式反应(Real-time PCR)检测肿瘤组织Beclin1,Atg5 mRNA转录水平。结果:芪玉三龙汤具有温和的移植瘤抑制作用,抑瘤率为31.2%;镜下观察芪玉三龙汤组肿瘤组织可见片状坏死的肿瘤细胞;免疫组化及Western blot实验表明,与模型组比较,芪玉三龙汤能够上调Beclin1,Atg5,LC3B蛋白的表达(P<0.01),并且能促进LC3B-Ⅰ转化为LC3B-Ⅱ;Real-time PCR结果表明,与模型组比较,芪玉三龙汤能够促进Beclin1,Atg5 mRNA的转录(P<0.01)。结论:芪玉三龙汤对肺肿瘤生长具有温和地抑制作用,其作用机制可能与上调自噬关键分子Beclin1,Atg5,LC3B表达,促进LC3B-Ⅰ向LC3B-Ⅱ转化有关。Objective:To study the effect of Qiyu Sanlong decoction on the growth of subcutaneous tumor in lung cancer mice and the expressions of key autophagy molecule,yeast Atg6 homologous(Beclin1),autophagy related genes5(Atg5),and microtubule-associated protein1 light chain3(LC3B).Method:Lewis lung carcinoma cells(LLC)were used to reproduce the lung cancer mice transplanted model.After the modeling,the mice were randomly divided into model group,Qiyu Sanlong decoction group,chemotherapy group and combination group,with 18 transplanted mice in each group.In model group,mice were fed with 0.9%saline 20 mL·kg^-1 daily.In Qiyu Sanlong decoction group,mice were fed with Qiyu Sanlong decoction 80.48 g·kg^-1daily.The chemotherapy group was intraperitoneally injected with 0.4 mL cisplatin solution(DDP)at the 1st,3rd and 5th day.The combination group was orally given the drugs at the concentration of 80.48 g·kg^-1,and 0.4 mL DDP solution was intraperitoneally injected at the 1st,3rd and 5th day.After 21 days of continuous treatment,tumor tissue was exfoliated and weighed,and the tumor inhibition rate was calculated.Hematoxylin-eosin(HE)staining was used to observe the histological changes of tumor.The expressions and localizations of Beclin1 and LC3B in tumor tissues were detected by immunohistochemical staining.Protein expressions of Beclin1,Atg5,LC3B-Ⅰand LC3B-Ⅱwere determined by Western blot,and the ratio of LC3B-Ⅱ/LC3B-Ⅰwas calculated.The transcription levels of Beclin1,Atg5 mRNA in tumor tissues were detected by Real-time PCR.Result:Qiyu Sanlong decoction had a mild inhibitory effect on transplanted tumor,with an inhibitory rate of 31.2%.Under microscope,patchy necrotic tumor cells were observed in the tumor tissues of Qiyu Sanlong decoction group.Immunohistochemical staining and Western blot analysis showed that Qiyu Sanlong decoction could up-regulate the expressions of Beclin1,Atg5 and LC3B protein(P<0.01),and promote the conversion from LC3B-Ⅰinto LC3-Ⅱcompared with the model group.Real-time PCR results s

关 键 词：芪玉三龙汤 肺癌 自噬 自噬关键分子酵母Atg6同系物(Beclin1) 自噬相关基因5(Atg5) 微管相关蛋白1轻链3(LC3B) 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学]