基于自噬通路抑制PD-L1研究黄芩苷对A549细胞杀伤作用机制  

Mechanism of baicalin anti-A549 cells by inhibiting PD-L1 based on autophagy pathway

作　　者：王泽苑 王宇航 刘晓嘉 赵薇 张瑜[2] 鄢丹 WANG Ze-yuan;WANG Yu-hang;LIU Xiao-jia;ZHAO Wei;ZHANG Yu;YAN Dan(College of Pharmacy,Chengdu University of Traditional Chinese Medicine,Chengdu 611137,China;Beijing Institute of Clinical Pharmacy,Beijing Friendship Hospital,Capital Medical University,Beijing 100050,China)

机构地区：[1]成都中医药大学药学院,四川成都611137 [2]首都医科大学附属北京友谊医院,北京市临床药学研究所,北京100050

出　　处：《药学学报》2025年第3期747-754,共8页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金项目(82304776,82304512)。

摘　　要：阻断程序性死亡受体1/程序性死亡配体1 (programmed cell death protein 1/programmed cell death ligand 1,PD-1/PD-L1)为代表的肿瘤免疫疗法在肺癌治疗上获得较好的治疗效果,研发小分子药物阻断PD-1/PD-L1为肺癌免疫疗法提供新策略。本研究应用体外细胞模型探讨了黄芩苷(baicalin)对A549肺癌细胞PD-L1的表达调控及作用机制。利用CCK8实验检测黄芩苷对A549细胞活力影响;Western blot检测A549细胞PD-L1蛋白的表达情况;利用凋亡、自噬等小分子抑制剂挖掘黄芩苷细胞毒性的机制及其对PD-L1蛋白表达的相关性;透射电子显微镜下观察黄芩苷处理A549细胞后自噬囊泡的形成;荧光显微镜下观察A549给药黄芩苷后酸性自噬泡的改变。CCK8实验结果显示,黄芩苷(12.5~400μmol·L-1)可呈剂量依赖性抑制A549细胞增殖;经干扰素-γ (interferon-γ, IFN-γ)干扰后PD-L1蛋白表达的升高可随黄芩苷浓度的增加而降低;另一方面,自噬抑制剂wortmannin和chloroquine处理可回调黄芩苷引起的细胞毒性,透射电子显微镜及荧光显微镜下观察黄芩苷处理A549细胞后自噬囊泡数量增加, Western blot结果显示,黄芩苷可促进自噬相关蛋白LC3-II/I和beclin-1的表达;wortmannin干预后,黄芩苷引起的A549细胞酸性自噬泡数量减弱,自噬蛋白LC3-II/I的表达受到抑制,对PD-L1的抑制作用得到回调。上述结果表明,黄芩苷可能通过激活A549细胞中自噬蛋白LC3降低PD-L1的表达发挥抗肿瘤作用。本研究为黄芩苷开发为PD-1/PD-L1信号通路小分子阻断剂奠定了基础。Tumor immunotherapy represented by blocking programmed cell death protein 1/programmed cell death ligand 1(PD-1/PD-L1)has gained better therapeutic effect in lung cancer treatment,and the development of small molecule drugs to block PD-1/PD-L1 provides a new strategy for lung cancer immunotherapy.In this study,we applied an in vitro cell model to investigate the regulation of PD-L1 expression and the mechanism of action of baicalin on A549 lung cancer cells.The effects of baicalin on the viability of A549 cells were detected by CCK8assay;the expression of PD-L1 protein in A549 cells was detected by Western blot;the mechanism of baicalin cytotoxicity and its correlation with PD-L1 protein expression were investigated by using small-molecule inhibitors of apoptosis and autophagy;the formation of autophagic vesicles in A549 cells treated with baicalin was observed under transmission electron microscope.And the alterations of acid autophagic vesicles and autophagic lysosomes were observed under fluorescence microscope.The results of CCK8 experiments showed that baicalin(12.5-400μmol·L-1)inhibited the proliferation of A549 cells in a dose-dependent manner;at the same time,the elevation of PD-L1 protein expression after interferon-γ(IFN-γ)interference could be reduced with the increase of baicalin concentration;on the other hand,treatment with autophagy inhibitors,wortmannin,and chloroquine,could call back the baicalin-induced cytotoxicity,transmission electron microscopy and fluorescence microscopy showed that the number of autophagic vesicles increased after baicalin treatment of A549 cells,and Western blot results showed that baicalin promoted the expression of autophagy-related proteins LC3-II/I and beclin-1;the number of baicalininduced acidic autophagic vesicles in A549 cells was attenuated after the intervention of wortmannin,and at the same time the LC3-II/I expression was inhibited and the inhibitory effect on PD-L1 was regulated.The above results suggest that baicalin may exert antitumor effects by activa

关 键 词：黄芩苷 非小细胞肺癌细胞A549 程序性死亡配体1 自噬 

分 类 号：R917[医药卫生—药物分析学]