肺岩宁方调控EMT逆转非小细胞肺癌顺铂耐药  

Chinese Herbal Medicine Feiyanning Prescription Regulates EMT to Reverse Cisplatin Resistance in Non-small Cell Lung Cancer

作　　者：夏尽力 蔡玥娇[1] 吴新鸿 王中奇[1] 邓海滨[1] XIA Jinli;CAI Yuejiao;WU Xinhong;WANG Zhongqi;DENG Haibin(Longhua Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China)

机构地区：[1]上海中医药大学附属龙华医院,上海200032

出　　处：《中国实验方剂学杂志》2023年第4期43-51,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：上海市教育委员会科研创新计划重大项目(2017-01-07-00-10-E00064);上海市中医药领军人才培养计划项目(ZY(2018-2020)-RCPY-10010)。

摘　　要：目的:观察肺岩宁方(肺岩宁)对非小细胞肺癌顺铂耐药的影响,并探究其可能存在的机制。方法:细胞增殖与活性检测(CCK-8)法观察顺铂(0、2.0、4.0、6.0、8.0、10.0 mg·L^(-1))对A549和A549/DDP(顺铂耐药)细胞细胞增殖能力的影响,肺岩宁(0、100、200、300、400、500、600 mg·L^(-1))对A549/DDP细胞增殖能力影响;免疫荧光染色、蛋白免疫印迹法(WB)观察A549和A549/DDP组上皮间质转化(EMT)相关蛋白表达;将A549/DDP细胞分为空白组、肺岩宁组(200 mg·L^(-1))、顺铂组(6.0 mg·L^(-1))和联合用药组[肺岩宁(200 mg·L^(-1))+顺铂(6.0 mg·L^(-1))],并进行相应处理,侵袭小室(transwell)实验比较各组侵袭能力;WB检测各组EMT相关蛋白表达;实时荧光定量聚合酶链式反应(Real-time PCR)和免疫荧光染色分别检测各组耐药因子mRNA和蛋白表达。结果:与A549组比较,A549/DDP组对顺铂耐药性显著增高(P<0.01);E-钙黏蛋白(E-cadherin)蛋白表达降低,波形蛋白(Vimentin)、N-钙黏蛋白(N-cadherin)、锌指转录因子(Snail)蛋白表达升高(P<0.05,P<0.01)。经肺岩宁、顺铂处理后,与空白组比较,肺岩宁可浓度依赖性抑制A549/DDP细胞增殖(P<0.01);肺岩宁组、顺铂组、联合用药组侵袭能力降低(P<0.01);与顺铂组比较,联合用药组侵袭能力显著降低(P<0.01)。与空白组比较,肺岩宁组E-cadherin蛋白表达升高,N-cadherin、Vimentin、Snail蛋白表达降低(P<0.01),顺铂组E-cadherin、N-cadherin、Vimentin蛋白表达降低,Snail蛋白表达升高(P<0.05,P<0.01);联合用药组E-cadherin、N-cadherin、Vimentin、Snail蛋白表达降低(P<0.01);与顺铂组比较,联合用药组E-cadherin蛋白表达升高,N-cadherin、Vimentin、Snail蛋白表达降低(P<0.01)。与空白组比较,肺岩宁组肺耐药相关蛋白(LRP)、多药耐药因子1(MDR1)蛋白和mRNA表达降低,拓扑异构酶Ⅱα(TOPOⅡα)蛋白和mRNA表达升高(P<0.01);顺铂组LRP、MDR1、TOPOⅡα蛋白和mRNA表达升高(P<0.01);联合用药�Objective:To observe the effect of Feiyanning prescription(FYN)on cisplatin(DDP)resistance in non-small cell lung cancer(NSCLC)and explore the underlying mechanism.Method:Cell counting kit-8(CCK-8)assay was used to detect the proliferation of A549 and A549/DDP(DDP-resistant)cells treated by DDP(0,2.0,4.0,6.0,8.0,10.0 mg·L-1)and the proliferation of A549/DDP cells treated by FYN(0,100,200,300,400,500,600 mg·L^(-1)).Based on immunofluorescence staining and Western blot(WB),the expression of epithelial mesenchymal transition(EMT)-related proteins in A549 and A549/DDP groups was observed.A549/DDP cells were classified into control group,FYN group(200 mg·L^(-1)),DDP group(6.0 mg·L^(-1)),and combination group[FYN(200 mg·L^(-1))+DDP(6.0 mg·L^(-1))]and respectively treated with corresponding drugs.Then,invasion ability of each group was examined by transwell assay,and the expression of EMT-related proteins in each group by WB.Moreover,real-time fluorescence quantitative polymerase chain reaction(Realtime PCR)and immunofluorescence staining were separately applied to detect the mRNA and protein expression of drug resistance-related factors in each group,respectively.Result:Compared with A549 group,A549/DDP group showed high resistance to DDP(P<0.01),low expression of E-cadherin,and high protein expression of Vimentin,N-cadherin,and Snail(P<0.05,P<0.01).As compared with the control group,FYN inhibited the proliferation of A549/DDP cells in a concentration-dependent manner(P<0.01),and the FYN group,DDP group,and combination group demonstrated low invasion ability(P<0.01).In addition,the invasion ability in the combination group was particularly lower than that in the DDP group(P<0.01).The expression of E-cadherin protein was higher and the protein expression of N-cadherin,Vimentin,and Snail was lower in the in FYN group than in the control group(P<0.01).The protein expression of E-cadherin,N-cadherin,and Vimentin was lower and the expression of Snail was higher in the DDP group than in the control group(P<0.05,P<0.01)

关 键 词：肺岩宁 非小细胞肺癌 耐药 上皮间质转化 顺铂 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学] R2-031R285.5