补中益气汤通过Nrf2/ROS通路调控线粒体途径细胞凋亡改善A549/DDP细胞顺铂耐药的分子机制  

Buzhong Yiqitang Regulates Apoptosis via Nrf2/ROS Pathway to Alleviate Cisplatin Resistance in A549/DDP Cells

作　　者：牟琪瑞 李贺[1] 刘玥彤 高原[1] 刘春英[1] MU Qirui;LI He;LIU Yuetong;GAO Yuan;LIU Chunying(College of Integrative Chinese and Western Medicine,Liaoning University of Traditional Chinese Medicine,Shenyang 110847,China)

机构地区：[1]辽宁中医药大学中西医结合学院,沈阳110847

出　　处：《中国实验方剂学杂志》2024年第1期95-102,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(81973735);辽宁省教育厅科学研究经费面上项目(LJKZ0901)。

摘　　要：目的:探讨补中益气汤含药血清通过核因子E_2相关因子2(Nrf2)/活性氧(ROS)通路调控线粒体途径细胞凋亡改善人非小细胞肺癌顺铂耐药细胞(A549/DDP)顺铂耐药的机制。方法:制备补中益气汤含药血清及培养A549/DDP细胞,并进行随机分组为空白组(10%空白血清)、顺铂组(10%空白血清+20 mg·L^(-1)顺铂)、补中益气汤组(10%含药血清+20 mg·L^(-1)顺铂)、ML385组(10%空白血清+5μmol·L^(-1)ML385+20 mg·L^(-1)顺铂)、补中益气汤联合ML385组(10%含药血清+5μmol·L^(-1)ML385+20 mg·L^(-1)顺铂)、叔丁基对苯二酚(TBHQ)组(10%空白血清+5μmol·L^(-1)TBHQ+20 mg·L^(-1)顺铂)、补中益气汤联合TBHQ组(10%含药血清+5μmol·L^(-1)TBHQ+20 mg·L^(-1)顺铂),应用细胞增殖与活性检测法(CCK-8)检测各组顺铂半数抑制浓度(IC_(50))值并计算耐药指数(RI),流式细胞术检测各组细胞凋亡率,DCFH-DA荧光探针法检测各组活性氧(ROS)含量,蛋白免疫印迹法(Western blot)检测各组Nrf2、活化的胱天蛋白酶-3(cleaved Caspase-3)、细胞色素C(CytC)、B细胞淋巴瘤-2(Bcl-2)蛋白表达。结果:与顺铂组比较,补中益气汤组、ML385组、补中益气汤联合ML385组A549/DDP细胞顺铂的IC_(50)及RI均明显降低(P<0.05);与空白组比较,顺铂组、补中益气汤组、ML385组、补中益气汤联合ML385组A549/DDP细胞的凋亡率均明显升高(P<0.05);与空白组比较,顺铂组细胞内Nrf2表达明显升高(P<0.05),与顺铂组比较,补中益气汤组、ML385组、补中益气汤联合ML385组Nrf2表达均明显降低(P<0.05),上述各组ROS表达量均明显升高(P<0.05),cleaved Caspase-3、CytC蛋白表达均升高、Bcl-2蛋白表达明显降低(P<0.05),其中以补中益气汤联合ML385组效果最为明显。与顺铂组比较,TBHQ组顺铂IC_(50)值及RI明显升高(P<0.05),A549/DDP细胞凋亡率明显降低(P<0.05),Nrf2蛋白表达明显升高(P<0.05),ROS表达下降(P<0.05),cleaved Caspase-3、CytC蛋白表达降低、Bcl-2蛋白表达明显Objective:To explore the mechanism of Buzhong Yiqitang-containing serum in alleviating the cisplatin resistance in human non-small cell lung cancer(A549/DDP)cells via regulating the nuclear factor E2-related factor 2(Nrf2)/reactive oxygen species(ROS)signaling pathway.Method:The serum containing Buzhong Yiqitang was prepared and A549/DDP cells were cultured and randomly grouped:blank(10%blank serum),cisplatin(10%blank serum+20 mg·L^(-1) cisplatin),Buzhong Yiqitang(10%Buzhong Yiqitangcontaining serum+20 mg·L^(-1) cisplatin),ML385(10%blank serum+5μmol·L^(-1) ML385+20 mg·L^(-1) cisplatin),Buzhong Yiqitang+ML385(10%Buzhong Yiqitang-containing serum+5μmol·L^(-1) ML385+20 mg·L^(-1) cisplatin),tertiary butylhydroquinone(TBHQ)(10%blank serum+5μmol·L^(-1) TBHQ+20 mg·L^(-1) cisplatin),and Buzhong Yiqitang+TBHQ(10%Buzhong Yiqitang-containing serum+5μmol·L^(-1) TBHQ+20 mg·L^(-1) cisplatin).The median inhibitory concentration(IC_(50))of cisplatin in each group was determined by the cell counting kit-8(CCK-8)method and the resistance index(RI)was calculated.The apoptosis rate was detected by flow cytometry.The ROS content of each group was determined with the DCFH-DA fluorescence probe.Western blot was employed to determine the protein levels of Nrf2,cleaved cysteinyl aspartate-specific protease-3(cleaved Caspase-3),cytochrome C(Cyt C),and B-cell lymphoma-2(Bcl-2).Result:Compared with those in the cisplatin group,the IC_(50) and RI of A549/DDP cells to cisplatin in Buzhong Yiqitang,ML385,and Buzhong Yiqitang+ML385 groups decreased(P˂0.05).Compared with the blank group,the cisplatin,Buzhong Yiqitang,ML385,and Buzhong Yiqitang+ML385 groups showed increased apoptosis rate of A549/DDP cells(P˂0.05).Compared with the blank group,cisplatin promoted the expression of Nrf2(P˂0.05).Compared with the cisplatin group,Buzhong Yiqitang,ML385,and Buzhong Yiqitang+ML385 inhibited the expression of Nrf2(P<0.05),elevated the ROS level(P˂0.05),up-regulated the protein levels of cleaved Caspase-3 and Cyt C,and down-regulated th

关 键 词：补中益气汤 非小细胞肺癌 核因子E_(2)相关因子2(Nrf2)/活性氧(ROS) 线粒体途径细胞凋亡 顺铂耐药 

分 类 号：R2-0[医药卫生—中医学] R22R242R285.5R734.2