清燥救肺汤调控AMPK通路诱导Lewis荷瘤小鼠肺癌细胞自噬体膜及溶酶体形成的机制探讨  

The Mechanism of Qingzao Jiufei Decoction Regulatingg AMPK Pathway Inducing the Formation of Autophagosome Membrane and Lysosome in LunggCancerCells of Lewis Tumor-Bearing Mouse

作　　者：张汗顺 余功 郑鸿翔[1] 谢斌[1] ZHANG Hanshun;YU Gong;ZHENG Hongxiang;XIE Bin(Jiangxi University of Chinese Medicine,Nanchang 330004 Jiangxi,China)

机构地区：[1]江西中医药大学,江西南昌330004

出　　处：《中药新药与临床药理》2023年第9期1195-1202,共8页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：江西省自然科学基金项目(20202BAB206075);江西省教育厅科技项目(GJJ201202);江西省一流学科建设科研启动基金专项(JXSYLXK-ZHYI059);江西省中医药管理局中医药科技计划项目(2020A0325);2021年度江西中医药大学校级研究生创新专项资金项目(JZYC21S64)。

摘　　要：目的基于腺苷酸活化蛋白激酶(AMPK)通路探讨清燥救肺汤诱导Lewis荷瘤小鼠肺癌细胞自噬体膜及溶酶体形成的机制。方法将50只雄性C57BL/6J小鼠随机分为模型组、环磷酰胺(CTX)组(50 mg·kg^(-1))、清燥救肺汤组(11 g·kg^(-1))、AMPK抑制剂组(Compound C,10 mg·kg^(-1))及清燥救肺汤+AMPK抑制剂组(11 g·kg^(-1)清燥救肺汤+10 mg·kg^(-1)Compound C)。各组小鼠均右腋皮下注射Lewis肺癌细胞构建肺癌荷瘤模型。清燥救肺汤组和清燥救肺汤+AMPK抑制剂组需造模前14 d开始灌胃清燥救肺汤(11 g·kg^(-1)·d^(-1))。造模24 h后,CTX组以50 mg·kg^(-1)CTX隔日腹腔注射1次,共7次;AMPK抑制剂组和清燥救肺汤+AMPK抑制剂组均腹腔注射Compound C(10 mg·kg^(-1)·d^(-1)),清燥救肺汤组和清燥救肺汤+AMPK抑制剂组继续按照设定剂量灌胃清燥救肺汤(11 g·kg^(-1)·d^(-1)),连续14 d。采用Western Blot法检测肺癌组织自噬体膜形成相关蛋白[自噬关键分子酵母ATG6同系物(Beclin-1)、磷酸化自噬关键分子酵母ATG6同系物(p-Beclin-1)、Ⅲ型磷脂酰肌醇3-激酶(VPS34)、磷酸化Ⅲ型磷脂酰肌醇3-激酶(p-VPS34)]的表达水平;RT-q PCR法检测肺癌组织中自噬相关蛋白9A(ATG9A)、乙酰辅酶A合成酶2(ACSS2)、转录因子增强子3(TFE3)m RNA表达水平;免疫荧光双重染色法检测肺癌组织中自噬蛋白微管相关蛋白轻链3B(LC3B)及P62蛋白表达情况。结果与模型组比较,清燥救肺汤组和CTX组小鼠肺癌组织中Beclin-1、p-Beclin-1蛋白表达水平及p-Beclin-1/Beclin-1比值显著升高(P<0.05,P<0.01),VPS34、p-VPS34蛋白表达水平及p-VPS34/VPS34比值均显著升高(P<0.05,P<0.01),ATG9A、ACSS2 m RNA表达水平显著升高(P<0.05,P<0.01),TFE3 m RNA表达水平显著降低(P<0.01),LC3B蛋白荧光强度表达水平显著升高(P<0.01),P62蛋白荧光强度表达水平显著降低(P<0.01)。与清燥救肺汤组比较,清燥救肺汤+AMPK抑制剂组小鼠肺癌组织中p-Beclin-1蛋白表达水平及pObjectivee To investigate the mechanism of Qingzao Jiufei Decoction on the formation of autophagosome membrane and lysosome in lung cancer cells of Lewis tumor-bearing mouse based on AMPK pathway.Methods Fifty male C57BL/6J mice were randomly divided into model group,cyclophosphamide(CTX)group(50mg·kg^(-1)),Qingzao Jufei Decoction group(11g·kg^(-1)),AMPK inhibitor group(Compound C,10mg·kg^(-1)),Qingzao Jiufei Decoction+AMPK inhibitor group(11 g·kg^(-1)Qingzao Jiufei Decoction+10 mg·kg^(-1)Compound C).Lewis lung cancer cells were subcutaneously injected into the right axilla to construct a tumor-bearing model.Qingzao Jiufei Decoction group and Qingzao Jiufei Decoction+AMPK inhibitor group were required to start administration of Qingzao Jiufei Decoction 14 days before modeling(11 g·kg^(-1)·d^(-1)).After 24 hours of modeling,CTX group was intraperitoneally injected once every other day for 7 times in total.AMPK inhibitor group and Qingzao Jiufei Decoction+AMPK inhibitor group were intraperitoneally injected Compound C(10 mg·kg^(-1)·d^(-1)).Qingzao Jiufei Decoction group and Qingzao Jiufei Decoction+AMPK inhibitor group continued to be given Qingzao Jiufei Decoction(11 g·kg^(-1)·d^(-1))according to the set dose for 14 consecutive days.Western Blot assay was used to detect the expression level of autophagosomal membrane formation related proteins in lung cancer[the expressions of autophagy key molecule yeast ATG6(Beclin-1),phosphorylated autophagy key molecule yeast ATG6(p-Beclin-1),typeⅠphosphatidylinositol 3-kinase(VPS34)and phosphorylated typeⅡphosphatidylinositol 3-kinase(p-VPS34)];the mRNA expressions of autophagy-associated protein 9A(ATG9A),acetyl-CoA synthetase 2(ACSS2)and transcription factor enhancer 3(TFE3)were detected by RT-qPCR;double immunofluorescence staining was used to detect the protein expressions of microtubule-associated protein light chain 3B(LC3B)and P62.Results Compared with the model group,the protein expressions of Beclin-1,p-Beclin-1,VPS34,p-VPS34 and the ratio of p-Beclin-

关 键 词：清燥救肺汤 肺癌 腺苷酸活化蛋白激酶通路 自噬体膜 溶酶体 小鼠 

分 类 号：R285.5[医药卫生—中药学]