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p-Beclin-1/Beclin-1比值显著降低(P<0.05,P<0.01),VPS34、p-VPS34蛋白表达水平及p-VPS34/VPS34比值均显著降低(P<0.05,P<0.01),ATG9A、ACSS2 m RNA表达水平显著降低(P<0.05,P<0.01),TFE3 m RNA表达水平显著上升(P<0.01),LC3B蛋白荧光强度表达水平显著降低(P<0.01),P62蛋白荧光强度表达水平明显升高(P<0.05)。与AMPK抑制剂组比较,清燥救肺汤+AMPK抑制剂组小鼠肺癌组织中Beclin-1、p-Beclin-1蛋白表达水平显著升高(P<0.01);VPS34、p-VPS34蛋白表达水平及p-VPS34/VPS34比值均显著升高(P<0.01);ATG9A、ACSS2 m RNA表达水平显著升高(P<0.05,P<0.01)。结论清燥救肺汤诱导肺癌细胞自噬体膜及溶酶体形成的机制可能是通过激活Beclin-1、VPS34蛋白,上调ATG9A、ACSS2 m RNA表达,抑制TFE3 mRNA表达实现的。
Objectivee 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-1/Beclin-1 and p-VPS34/VPS34 in Qingzao Jiufei Decoction group were increased(P<0.01,P<0.05).The mRNA expressions of ATG9A and ACSS2 were increased(P<0.01,P<0.05),the mRNA expression of TFE3 was decreased(P<0.01),the immunofluorescence intensity of LC3B was increased(P<0.05),and the immunofluorescence intensity of P62 protein expression level was decreased(P<0.01);compared with Qingzao Jiufei Decoction group,the protein expressions of p-Beclin-1,VPS34,p-VPS34 and the ratio of p-Beclin-1/Beclin-1 and p-VPS34/VPS34 in Qingzao Jiufei Decoction+AMPK inhibitor group were decreased(P<0.01,P<0.05).The mRNA expression levels of ATG9A and ACSS2 were decreased(P<0.01,P<0.05),the mRNA expression of TFE3 was increased(P<0.01),the immunofluorescence intensity of LC3B protein was decreased(P<0.01),and the immunofluorescence intensity of P62 protein was increased(P<0.05).Compared to the AMPK inhibitor group,the protein expression levels of Beclin-1 and p-Beclin-1 in lung cancer tissues of mice in Qingzao Jiufei Decoction+AMPK inhibitor group were significantly increased(P<0.01),protein expression levels of VPS34,p-VPS34 and p-VPS34/VPS34 ratio were significantly increased(P<0.01),mRNA expression levels of ATC9A and ACSS2 were significantly increased(P<0.01,P<0.05).Conclusion The mechanism of Qingzao Jiufei Decoction induced the formation of autophagosome membrane and lysosome in lung cancer cells may be achieved by activating the protein expressions of Beclin-1 and VPS34,up-regulating the mRNA expressions of ATG9A and ACSS2,and inhibiting the mRNA expression of TFE3.
作者
张汗顺
余功
郑鸿翔
谢斌
ZHANG Hanshun;YU Gong;ZHENG Hongxiang;XIE Bin(Jiangxi University of Chinese Medicine,Nanchang 330004 Jiangxi,China)
出处
《中药新药与临床药理》
CAS
CSCD
北大核心
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)。
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
Qingzao Jiufei Decoction
lung cancer
adenylate activates protein kinase pathway
autophagosome membrane
lysosome
mouse
