归芪益元膏通过调控ERK信号通路增加自噬通量稳定PD-L1的表达对Lewis肺癌小鼠的影响  

Effect of Guiqi Yiyuan Ointment on Lewis Lung Cancer Mice by Increasing Autophagic Flux and Stabilizing PD-L1 Expression Through Regulation of ERK Signaling Pathway

作　　者：杨楠 马抢平 梁建庆 苗珂君 李尚 李金田[2,3] 李娟 YANG Nan;MA Qiangping;LIANG Jianqing;MIAO Kejun;LI Shang;LI Jintian;LI Juan(School of Basic Medicine,Gansu University of Chinese Medicine,Lanzhou 730101,China;The KeyLaboratory of the Ministry of Education of Dunhuang Medicine and Transformation,Lanzhou 730000,China;Clinical College of Chinese Medicine,Gansu University of Chinese Medicine,Lanzhou 730101,China)

机构地区：[1]甘肃中医药大学基础医学院,兰州730101 [2]敦煌医学与转化教育部重点实验室,兰州730000 [3]甘肃中医药大学中医临床学院,兰州730101

出　　处：《中国实验方剂学杂志》2025年第8期107-114,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(82160872);甘肃省教育厅“双一流”科研重点项目(GSSYLXM-05);甘肃省中医药研究中心开放课题(zyzx-2020-zx17);2022年度中医学一级学科“岐黄英才”导师专项(ZYXKBD-202202,ZYXKSD-202211);敦煌医学与转化教育部重点实验室开放课题(DHYX22-06)。

摘　　要：目的:基于细胞外调节蛋白激酶(ERK)信号通路探讨归芪益元膏对Lewis肺癌小鼠的抑瘤作用及机制。方法:复制Lewis肺癌小鼠模型,除空白组外,将模型鼠随时分为模型组、归芪益元膏低、中、高剂量组和细胞外调节蛋白激酶1/2(ERK1/2)抑制剂组,每组10只,归芪益元膏低、中、高剂量组分别给予归芪益元膏(1.75、3.5、7.0 g·kg^(-1)·d^(-1))灌胃,抑制剂组给予ERK1/2抑制剂LY3214996(100 mg·kg^(-1)·d^(-1))灌胃,连续14 d后取材。苏木素-伊红(HE)染色观察小鼠肿瘤组织病理变化,透射电镜观测肿瘤组织细胞超微结构的变化,免疫荧光测定肿瘤组织磷酸化细胞外调节蛋白激酶1/2(p-ERK1/2)、程序性细胞死亡配体-1(PD-L1)蛋白表达,蛋白免疫印迹法(Western blot)及实时荧光定量聚合酶链式反应(Real-time PCR)检测肿瘤组织p-ERK1/2、PD-L1、自噬标志蛋白Beclin-1、自噬相关蛋白p62和微管相关蛋白1轻链3(LC3)Ⅰ、LC3Ⅱ蛋白、mRNA表达。结果:与模型组比较,归芪益元膏低、中剂量组平均瘤重降低(P<0.05),归芪益元膏高剂量组、抑制剂组平均瘤质量显著降低(P<0.01);各组肿瘤细胞逐渐不规则,细胞核破裂,细胞崩解及坏死区域扩大;各组肿瘤组织细胞器消融逐渐增多且自噬小体数量逐渐增加;归芪益元膏低、中剂量组p-ERK1/2和PD-L1平均荧光强度降低(P<0.05),归芪益元膏高剂量组、抑制剂组p-ERK1/2和PD-L1平均荧光强度显著降低(P<0.01);归芪益元膏中剂量组ERK1/2、PD-L1、Beclin-1、p62 mRNA表达明显降低(P<0.05),LC3Ⅰ/ⅡmRNA表达升高(P<0.05),归芪益元膏高剂量组、抑制剂组ERK1/2、PD-L1、Beclin-1、p62 mRNA表达显著降低(P<0.01),LC3Ⅰ/ⅡmRNA表达显著升高(P<0.01);归芪益元膏中剂量组p-ERK1/2、PD-L1、Beclin-1、p62蛋白表达降低(P<0.05),LC3Ⅰ/Ⅱ蛋白表达升高(P<0.05),归芪益元膏高剂量组、抑制剂组p-ERK1/2、PD-L1、Beclin-1、p62蛋白表达显著降低(P<0.01),LC3Ⅰ/Ⅱ蛋�Objective:To investigate the antitumor effect and mechanism of Guiqi Yiyuan ointment on Lewis lung cancer mice based on the extracellular regulatory protein kinase(ERK)signaling pathway.Methods:A Lewis lung cancer mouse model was established.Except for the blank group,the model mice were randomly divided into the model group,Guiqi Yiyuan ointment low,medium,and high dose groups,and the extracellular ERK1/2 inhibitor group,with 10 mice per group.The Guiqi Yiyuan ointment was administered by gavage at doses of 1.75,3.5,7.0 g·kg^(-1)·d^(-1) for the low,medium,and high dose groups,respectively.The ERK1/2 inhibitor group was given the ERK1/2 inhibitor LY3214996(100 mg·kg^(-1)·d^(-1))by gavage.The treatment was administered for 14 consecutive days,after which samples were collected.Tumor histopathological changes were observed using hematoxylin-eosin(HE)staining.Transmission electron microscopy was used to observe ultrastructural changes in tumor cells.Immunofluorescence was performed to measure the phosphorylation of ERK1/2(p-ERK1/2)and the expression of programmed cell death ligand^(-1)(PD-L1)in tumor tissues.Western blot and real-time quantitative polymerase chain reaction(Real time PCR)were used to detect the expression of p-ERK1/2,PD-L1,the autophagy marker Beclin-1,the autophagic protein p62,and the microtubule-associated protein light chains LC3Ⅰand LC3Ⅱat both the protein and gene levels.Results:Compared with the model group,the average tumor weight was significantly reduced in the low and medium dose groups of Guiqi Yiyuan ointment(P<0.05),and markedly reduced in the high dose and inhibitor groups(P<0.01).Tumor cells in all treatment groups became progressively irregular,with ruptured nuclei and expanded areas of cell disintegration and necrosis.The number of organellar ablations in tumor tissues increased,and the number of autophagic vesicles also increased in all groups.The mean fluorescence intensity of p-ERK1/2 and PD-L1 was reduced in the low and medium dose groups of Guiqi Yiyuan ointment(P<0.05)

关 键 词：肺癌 归芪益元膏 细胞外调节蛋白激酶(ERK)信号通路 自噬 程序性细胞死亡配体-1(PD-L1) 

分 类 号：R256[医药卫生—中医内科学] R285[医药卫生—中医学] R273