华蟾素调控AKT介导的上皮间质转化抑制肝细胞癌肺转移裸鼠模型的作用机制  

Mechanism of action of cinobufotalin in inhibiting lung metastasis of hepatocellular carcinoma by regulating AKTmediated epithelial-mesenchymal transition in a nude mouse model

作　　者：杨悦 续嗣钰 王珏 杜施霖 张春蕾 宋海燕 YANG Yue;XU Siyu;WANG Jue;DU Shilin;ZHANG Chunlei;SONG Haiyan;无(Institute of Digestive Diseases,Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China;Department of Emergency,Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China;Department of Emergency,Zhongshan Hospital,Fudan University,Shanghai 200032,China)

机构地区：[1]上海中医药大学附属龙华医院脾胃病研究所,上海200032 [2]上海中医药大学附属龙华医院急诊科,上海200032 [3]复旦大学附属中山医院急诊科,上海200032

出　　处：《临床肝胆病杂志》2024年第9期1840-1847,共8页Journal of Clinical Hepatology

基　　金：上海市青年科技英才扬帆计划项目(20YF1450000);龙华医院科技创新项目(CX202205);上海市卫生健康委卫生行业临床研究专项(202040223)。

摘　　要：目的研究华蟾素通过调控肝细胞癌(HCC)上皮间质转化(EMT)抑制HCC转移的作用和机制。方法将36只6周龄雄性BALB/c裸鼠尾静脉注射MHCC97H细胞建立肝癌肺转移瘤模型,随机分为华蟾素高、低剂量组和对照组。建模当日起分别腹腔注射华蟾素120μL/kg、60μL/kg或生理盐水,每周2次。8周后取肺组织行HE染色检测肝癌肺转移率。MHCC97H细胞用华蟾素高、低剂量(2.5μL/m L、5μL/m L)干预,通过划痕实验、RT-PCR以及Western Blot检测细胞迁移能力和EMT相关分子的表达。用CoCl2孵育模拟低氧环境诱导MHCC97H细胞,同时加入高、低剂量华蟾素干预,通过划痕实验和Western Blot检测华蟾素对低氧诱导的细胞迁移能力和EMT的影响。使用转录组学分析华蟾素对MHCC97H细胞的效应机制。用Western Blot检验华蟾素干预对MHCC97H细胞的蛋白激酶B(AKT)、磷酸化AKT(P-AKT)表达水平的影响。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验,两组间比较采用成组t检验。结果华蟾素干预组裸鼠较对照组肝癌肺转移率下降。与对照组相比,华蟾素干预使MHCC97H细胞划痕愈合率减小、上皮型分子表达上调(t=2.860,P<0.05),并使EMT转录因子和基质型分子下调(t值分别为3.545、2.022、2.852、2.341,P值均<0.05)。低氧诱导上调MHCC97H细胞划痕愈合率和基质型分子、EMT转录因子表达水平(P值均<0.05),华蟾素干预逆转EMT变化并抑制划痕愈合(P值均<0.05)。肝癌细胞转录组学分析显示,华蟾素组与对照组存在显著的基因差异,华蟾素主要影响了肿瘤、代谢、免疫和信号传导相关基因表达,其中AKT信号转导通路中的差异基因数量最多。进一步检测发现华蟾素干预可下调HCC细胞AKT、P-AKT和P-AKT/AKT的水平(t值分别为2.434、3.401、2.258,P值均<0.05)。结论华蟾素可抑制肝癌转移,尤其对于低氧环境诱导的肝癌转移具有显著抑制作用,调控AKT�Objective To investigate the effect and mechanism of cinobufotalin in inhibiting hepatocellular carcinoma(HCC)metastasis by regulating epithelial-mesenchymal transition(EMT).Methods A total of 36 male BALB/c nude mice,aged 6 weeks,were given injection of MHCC97H cells via the caudal vein to establish a model of HCC lung metastasis,and then the mice were randomly divided into high-and low-dose cinobufotalin groups and control group.Since the day of modeling,the mice in the high-and low-dose cinobufotalin groups were given intraperitoneal injection of cinobufotalin at a dose of 120μL/kg and 60μL/kg,respectively,and those in the control group were given intraperitoneal injection of normal saline,twice a week.After 8 weeks,HE staining was performed for lung tissue to measure the lung metastasis rate of HCC.MHCC97H cells were treated with high-dose(2.5μL/mL)or low-dose(5μL/mL)cinobufotalin for 24 hours,and wound healing assay,RT-PCR,and Western blot were used to measure cell migration ability and the expression of EMT-related molecules.MHCC97H cells were induced in a simulated hypoxic environment with CoCl2 incubation,with high-and low-dose cinobufotalin added for intervention,and wound healing assay and Western blot were used to investigate the effect of cinobufotalin on cell migration ability and EMT induced by hypoxia.Transcriptome analysis was used to investigate the effect mechanism of cinobufotalin on MHCC97H cells,and Western blot was used to observe the effect of cinobufotalin on the expression levels of protein kinase B(AKT)and phosphorylated AKT(P-AKT)in MHCC97H cells.A oneway analysis of variance was used for comparison of continuous data between multiple groups,and the least significant difference ttest was used for further comparison between two groups;the independent-samples t test was used for comparison of categorical data between two groups.Results Compared with the control group,the cinobufotalin group had a significant reduction in the lung metastasis rate of HCC.Compared with the control group,

关 键 词：癌 肝细胞 肿瘤转移 华蟾素 上皮-间质转化 

分 类 号：R735.7[医药卫生—肿瘤]