车叶草苷调节RAS/RAF/MEK/ERK信号通路对肝细胞癌细胞活性及裸鼠移植瘤生长的影响  

Effects of Asperuloside on the Activity of Hepatocellular Carcinoma Cells and the Growth of Transplanted Tumors in Nude Mice by Regulating RAS/RAF/MEK/ERK Signaling Pathway

作　　者：张小龙 王军委 李岩 刘雪 刘峥[1] 张继光[2] ZHANG Xiaolong;WANG Junwei;LI Yan;LIU Xue;LIU Zheng;ZHANG Jiguang(Third Department of Oncology,Handan Central Hospital,Handan Hebei 056000,China)

机构地区：[1]邯郸市中心医院肿瘤三科,河北邯郸056000 [2]邯郸市中心医院介入科,河北邯郸056000

出　　处：《联勤军事医学》2024年第9期729-734,757,共7页Military Medicine of Joint Logistics

基　　金：河北省医学科学研究课题计划(20241159)。

摘　　要：目的探讨车叶草苷调节Ras蛋白(Ras protein,RAS)/Raf蛋白激酶(Raf protein kinase,RAF)/丝裂原激活蛋白激酶激酶(mitogen-activated protein kinase kinase,MEK)/细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)信号通路对肝细胞癌(hepatocellular carcinoma,HCC)细胞活性及裸鼠移植瘤生长的影响。方法体外培养人HCC-LM3以细胞计数试剂盒(cell counting kit 8,CCK-8)法筛选车叶草苷体外最佳作用浓度。将HCC-LM3细胞随机分为对照组(细胞正常培养,不作其他干预)、车叶草苷组(3 mmol/L车叶草苷进行干预)、ML-098组(0.5μmol/L RAS激活剂ML-098进行干预)、车叶草苷+ML-098组(车叶草苷3 mmol/L和RAS激活剂ML-098终浓度0.5μmol/L联合干预)。以CCK-8法、流式细胞术分别检测各组HCC-LM3细胞活性、凋亡率;Western blot检测各组HCC-LM3细胞增殖、凋亡相关蛋白表达及RAS/RAF/MEK/ERK信号通路相关蛋白表达。制备HCC-LM3裸鼠原位癌模型并随机分为对照组(5 ml/kg生理盐水)、车叶草苷低剂量组(25 mg/ml车叶草苷)、车叶草苷中剂量组(50 mg/ml车叶草苷)、车叶草苷高剂量组(100 mg/ml车叶草苷)。检测各组裸鼠肝体比、肿瘤长径。以Ki67免疫组织化学染色法检测各组裸鼠肿瘤细胞增殖指数;Western blot检测各组裸鼠肿瘤RAS/RAF/MEK/ERK信号通路相关蛋白表达。结果与对照组相比,车叶草苷组HCC-LM3细胞凋亡率、裂解凋亡蛋白酶3(Cleaved caspase-3)与B细胞淋巴瘤2相关X蛋白(Bcl-2 associated X protein,Bax)、含半胱氨酸的天冬氨酸蛋白水解酶9(cysteinyl aspartate specific proteinase 9,caspase-9)蛋白表达均升高(P均<0.05),细胞活性、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)与RAS蛋白表达、p-RAF/RAF、p-MEK/MEK、p-ERK/ERK均降低(P均<0.05);ML-098对HCC-LM3细胞各指标的作用与车叶草苷相反(P<0.05)。与车叶草苷组相比,车叶草苷+ML-098组HCC-LM3细胞凋亡率、Cleaved caspase-3与Bax、caspase-9蛋白表达Objective To investigate the effects of asperuloside on the activity of hepatocellular carcinoma(HCC)cells and the growth of transplanted tumors in nude mice regulating Ras protein(RAS)/Raf protein kinase(RAF)/mitogen-activated protein kinase kinase(MEK)/extracellular signal-regulated kinase(ERK)signaling pathway.Methods In vitro cultured human HCC-LM3 were screened for the best action concentration of asperuloside in vitro by cell counting kit 8(CCK-8).HCC-LM3 cells were randomly divided into control group(normal cell culture,no other intervention),asperuloside group(intervention with 3 mmol/L asperuloside),ML-098 group(intervention with 0.5μmol/L RAS activator ML-098),and asperuloside+ML-098 group(combined intervention with asperuloside 3 mmol/L and RAS activator ML-098 final concentration of 0.5μmol/L).The activity and apoptosis rate of HCC-LM3 cells in each group were detected by CCK-8 method and flow cytometry,respectively;the proliferation and apoptosis of HCC-LM3 cells and the expression of RAS/RAF/MEK/ERK signaling pathway-related proteins were detected by Western blot.The HCC-LM3 nude mice in situ cancer model was prepared and randomly divided into control group(5 ml/kg normal saline),low-dose group of asperuloside(25 mg/ml asperuloside),middle-dose group of asperuloside(50 mg/ml asperuloside),and high-dose group of asperuloside(100 mg/ml asperuloside).The liver-to-body ratio and tumor length of nude mice in each group were detected.The proliferation of tumor cells in nude mice in each group was detected by Ki67 immunohistochemical staining.The expression of proteins related to RAS/RAF/MEK/ERK signaling pathway in nude mice in each group was detected by Western blot.Results Compared with the control group,the apoptosis rate of HCC-LM3 cells,the expression of Cleaved caspase-3,Bcl-2 associated X protein(Bax),and cysteine containing aspartate specific protein 9(caspase-9)were all increased in the asperuloside group(all P<0.05),and the cell activity,the expression of proliferating cell nuclear antigen(PCN

关 键 词：车叶草苷 肝细胞癌 细胞活性 移植瘤 Ras蛋白/Raf蛋白激酶/丝裂原激活蛋白激酶激酶/细胞外信号调节激酶信号通路 

分 类 号：R735.7[医药卫生—肿瘤]