康艾注射液通过抑制PI3K/Akt/mTOR信号通路诱导细胞周期阻滞及自噬性死亡改善A549/DDP细胞顺铂耐药性的研究  

Kangai Injection Reverses Cisplatin Resistance through PI3K/Akt/mTOR Signaling Pathway Mediated Cell Cycle Arrest and Autophagic Death in A549/DDP Cells

作　　者：吕佳璐 周欢 刘文俊[3] 王建光 王淳[1] LYU Jialu;ZHOU Huan;LIU Wenjun;WANG Jianguang;WANG Chung(College of Integrative Chinese and Western Medicine of Liaoning University of Traditional Chinese Medicine,Shenyang 110847,China;Traditional Chinese Medicine Encephalopathy Bone Injury Ward of Chaoyang Central Hospital,Chaoyang 122000,China;Experimental Teaching Center of 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 [2]朝阳市中心医院,朝阳122000 [3]辽宁中医药大学实验教学中心,沈阳110847

出　　处：《世界科学技术-中医药现代化》2024年第12期3127-3135,共9页Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology

基　　金：国家自然科学基金委员会面上项目(82174254):培土生金法干预mTOR-Mfn2-PKM2信号通路靶向线粒体动力学-糖酵解调控网络,改善NSCLC顺铂耐药机制的研究,负责人:王淳;辽宁省科技计划联合计划应用基础研究项目(2023JH2/101700210):扶正固本中药重塑糖脂代谢时空网络表观遗传调控HIF1α/SLC7A11诱导的铁死亡改善肺腺癌吉非替尼耐药机制及应用研究,负责人:刘文俊;辽宁中医药大学自然科学类青年项目(2021LZY004):培土生金法调控肺癌小鼠肠道微生物群介导免疫功能的研究,负责人:王建光。

摘　　要：目的靶向细胞周期及细胞自噬,探讨康艾注射液(Kangai injection,KAI)通过PI3K/Akt/mTOR信号通路改善人肺腺癌A549/DDP细胞顺铂耐药性的分子机制。方法将A549/DDP细胞随机分为空白对照组、顺铂组(20μmol·L^(-1)cisplatin)及低、中、高剂量KAI联合顺铂组(15 mg·mL^(-1)、25 mg·mL^(-1)、50 mg·mL^(-1)KAI+20μmol·L^(-1)cisplatin)。CCK-8法检测细胞活力、流式细胞术检测细胞周期分布情况、免疫细胞化学法检测p53蛋白的表达情况、蛋白质免疫印迹法分别检测p53、自噬相关蛋白LC3Ⅱ及自噬通路关键分子(mTOR、p-mTOR、Akt、p-Akt)蛋白表达水平,并利用PI3K/Akt信号通路抑制剂LY294002进一步明确KAI改善顺铂耐药性的分子机制。结果与对照组相比,顺铂组及低、中、高剂量KAI联合顺铂组细胞存活率显著下降(P<0.05);与顺铂组相比,中、高剂量KAI联合顺铂组细胞周期S+G2/M期细胞比率升高(P<0.05),低、中、高剂量KAI联合顺铂组p53蛋白平均光密度值显著增加(P<0.05),中、高剂量KAI联合顺铂组自噬相关蛋白LC3Ⅱ表达水平升高(P<0.05)、信号通路相关蛋白mTOR及Akt磷酸化(p-mTOR、p-Akt)水平降低(均P<0.01);通路抑制剂实验结果显示,LY294002可有效抑制KAI联合顺铂诱导的LC3Ⅱ蛋白表达上调及细胞死亡(均P<0.05)。结论KAI联合顺铂通过抑制PI3K/Akt/mTOR信号通路,诱导细胞周期阻滞及细胞自噬性死亡,改善NSCLC顺铂耐药性。Objective Targeting the cell cycle and autophagy,Kangai injection(KAI)was investigated through PI3K/Akt/mTOR Signaling pathways improve the molecular mechanism of cisplatin resistance in human lung adenocarcinoma A549/DDP cells.Methods A549/DDP cells were randomly divided into control group,cisplatin group(20μmol·L^(-1) cisplatin),low concentration KAI combined with cisplatin(15 mg·mL^(-1) KAI+20μmol·L^(-1) cisplatin),medium concentration KAI combined with cisplatin(25mg·mL^(-1) KAI+20μmol·L^(-1) cisplatin)and high concentration KAI combined with cisplatin(50 mg·mL^(-1) KAI+20μmol·L^(-1)cisplatin).The survival rate of cells was detected by CCK-8 method,the cell cycle distribution was detected by flow cytometry,the expression of p53 protein was detected by immunocytochemistry,and the expression levels of protein p53、autophagyrelated protein LC3 and key molecules of autophagy pathway(m TOR,p-m TOR,Akt and p-Akt)were detected by Western blotting.Besides,the inhibitor of PI3K/Akt signaling pathway was used to further verify the molecular mechanism of KAI in reversing cisplatin in A549/DDP cells.Results Compared with the control group,cisplatin group and different concentrations of KAI combined with cisplatin groups effectively inhibited cell viability(P<0.05);compared with the cisplatin group,increased the cell proportion in S+G2/M phase(P<0.05),the mean integral absorbance of p53 protein(P<0.05),and the expression level of autophagy-related protein LC3Ⅱ(P<0.05),but decreased the expression levels of p-mTOR and p-Akt(both P<0.01).Moreover,LY294002,the inhibitor of PI3K/Akt signaling pathway,could effectively inhibit the up-regulation of LC3Ⅱ expression and cell death induced by KAI combined with cisplatin(both P<0.05).Conclusion KAI combined with cisplatin can induce cell cycle arrest and autophagic death by inhibiting PI3K/Akt/mTOR signaling pathway,and improve cisplatin resistance in NSCLC.

关 键 词：非小细胞肺癌 顺铂耐药 康艾注射液 周期阻滞 自噬 

分 类 号：R285.5[医药卫生—中药学]