归芪益元膏联合顺铂通过调控PERK/eIF2α/ATF4/CHOP信号通路对Lewis肺癌荷瘤小鼠肿瘤生长的抑制作用  

Guiqi Yiyuan Ointment combined with cisplatin inhibits tumor growth in Lewis lung carcinoma-bearing mice by regulating PERK/eIF2α/ATF4/CHOP signaling pathway

作　　者：杨楠 梁建庆 苗珂君 马抢平 李金田[2,3] 李娟 YANG Nan;LIANG Jian-qing;MIAO Ke-jun;MA Qiang-ping;LI Jin-tian;LI Juan(School of Basic Medicine,Gansu University of Chinese Medicine,Lanzhou 730000,China;Key Laboratory of Dunhuang Medicine,Ministry of Education,Lanzhou 730000,China;Clinical College of Traditional Chinese Medicine,Gansu University of Chinese Medicine,Lanzhou 730000,China)

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

出　　处：《中国中药杂志》2025年第6期1592-1600,共9页China Journal of Chinese Materia Medica

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”导师专项基金博导项目(ZYXKBD-202202);2022年度中医学一级学科“岐黄英才”导师专项基金硕导项目(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：基于蛋白激酶R样内质网激酶(PERK)/真核翻译起始因子2α激酶(eIF2α)/活化转录因子4(ATF4)/C/EBP同源蛋白(CHOP)信号通路探讨归芪益元膏联合顺铂对Lewis肺癌荷瘤小鼠的抑瘤作用及机制。将60只SPF级雄性C57BL/6小鼠,采用随机数字表法取10只小鼠作为空白组,其余成功复制Lewis肺癌荷瘤小鼠模型后,随机分为模型组、顺铂组(5 mg·kg^(-1))以及归芪益元膏低、中、高剂量(1.7、3.5、7.05 g·kg^(-1))+顺铂(5 mg·kg^(-1))组,每组10只,顺铂每7 d给药1次,归芪益元膏每日给药1次。连续干预14 d后麻醉处死,剥离小鼠脾脏、胸腺、瘤体,称重记录,计算脾脏指数、胸腺指数和抑瘤率。苏木素-伊红(HE)染色观察小鼠肿瘤组织病理学变化;透射电镜观察肿瘤细胞内质网形态变化;免疫荧光法检测肿瘤组织中磷酸化eIF2α(p-eIF2α)、ATF4蛋白表达;Western blot法检测肿瘤组织中磷酸化PERK(p-PERK)、p-eIF2α、ATF4、CHOP、B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)、细胞周期蛋白依赖激酶抑制因子1A(p21)和细胞周期蛋白D1(cyclinD1)蛋白表达;实时荧光定量PCR法检测肿瘤组织中PERK、eIF2α、ATF4、CHOP、Bax、Bcl-2、p21、cyclinD1 mRNA表达。结果显示,与空白组比较,模型组小鼠脾脏指数、胸腺指数均明显下降(P<0.05)。与模型组比较,顺铂组小鼠脾脏指数、胸腺指数明显下降(P<0.05),归芪益元膏中、高剂量+顺铂组小鼠脾脏指数、胸腺指数明显升高(P<0.05),各组瘤质量均明显下降(P<0.05);肿瘤细胞溶解、核破裂逐渐增多;粗面内质网囊膜间隙逐渐增宽;各给药组p-eIF2α和ATF4平均荧光强度明显增强(P<0.05),p-PERK/PERK、p-eIF2α/eIF2α、ATF4、CHOP、Bax和p21蛋白表达明显上调(P<0.05),Bcl-2、cyclinD1蛋白及mRNA表达明显下调(P<0.05),PERK、eIF2α、ATF4、CHOP、Bax和p21 mRNA表达明显上调(P<0.05)。与顺铂组比较,各联合用药组小鼠脾脏指数、胸腺指数明显升高(P<0.05);归�This study aims to investigate the anti-tumor effect and mechanism of Guiqi Yiyuan Ointment combined with cisplatin on Lewis lung carcinoma-bearing mice via the protein kinase RNA-like endoplasmic reticulum kinase(PERK)/eukaryotic translation initiation factor 2α(eIF2α)/activated transcription factor 4(ATF4)/C/EBP homologous protein(CHOP)signaling pathway.Sixty SPF-grade male C57BL/6 mice were selected and assigned into a blank group and a modeling group by the random number table method.After modeling of the Lewis lung carcinoma,the mice in the modeling group were randomized into model,cisplatin(5 mg·kg^(-1),once a week),and low-,medium-,and high-dose(1.7,3.5,and 7.05 g·kg^(-1),respectively,once a day)Guiqi Yiyuan Ointment+cisplatin(5 mg·kg^(-1))groups(n=10).After 14 days of continuous intervention,the spleen,thymus,and tumor samples of the mice were collected,weighed,and recorded,and the spleen index,thymus index,and tumor suppression rate were calculated.Hematoxylin-eosin(HE)staining was employed to observe the pathological changes in the tumor tissue.The morphological changes of the endoplasmic reticulum of tumor cells were observed by transmission electron microscopy.The positive expression of phosphorylated eIF2α(p-eIF2α)and ATF4 in the tumor tissue was detected by immunofluorescence.Western blot was employed to determine the protein levels of phosphorylated PERK(p-PERK),p-eIF2α,ATF4,CHOP,B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),cyclin-dependent kinase inhibitor 1A(p21),and cyclinD1 in the tumor tissue.Real-time fluorescent quantitative PCR was employed to determine the mRNA levels of PERK,eIF2α,ATF4,CHOP,Bax,Bcl-2,p21,and cyclinD1 in the tumor tissue.Compared with the blank group,the model group showed decreases in spleen index and thymus index(P<0.05).Compared with the model group,the cisplatin group showed decreases in spleen index and thymus index(P<0.05),and the medium-and high-dose Guiqi Yiyuan Ointment+cisplatin groups presented increases in spleen index and thymus index(P<0.

关 键 词：归芪益元膏 非小细胞肺癌 顺铂 内质网应激 信号通路 

分 类 号：R285.5[医药卫生—中药学]