冬凌草甲素逆转BEL-7402/5-FU化疗耐药作用的体内研究  

Oridonin A reverses the chemoresistance of BEL-7402/5-FU in vivo

作　　者：史国军 王永生 何国浓 叶兴涛 施航 陈如儿 SHI Guo-jun;WANG Yong-sheng;HE Guo-nong;YE Xing-tao;SHI Hang;CHEN Ru-er(Department of Oncology,Ningbo Hospital of Traditional Chi-nese Medicine,Ningbo,Zhejiang 315010,China)

机构地区：[1]浙江省宁波市中医院肿瘤科,宁波315010

出　　处：《浙江中西医结合杂志》2023年第10期888-892,908,共6页Zhejiang Journal of Integrated Traditional Chinese and Western Medicine

基　　金：浙江省宁波市自然科学基金(No.202003N4216);第四轮省级重点学科"十二五"中西医结合肿瘤学(No.甬卫发[2021]96号);第五批全国中医临床优秀人才研修项目(No.国中医药人教函[2022]1号)。

摘　　要：目的探讨冬凌草甲素逆转人肝癌耐药细胞株BEL-7402/5-FU裸鼠移植瘤耐药效应及机制。方法取4~6周龄BALB/c雌性裸小鼠40只,皮下接种BEL-7402/5-FU细胞,建立BEL-7402/5-FU裸鼠移植瘤耐药模型,按照随机数字表法分成八组,即模型组,冬凌草甲素高、中、低剂量组,5-FU组和5-FU+冬凌草甲素高、中、低剂量组,每组5只。分组当天开始灌胃给药,模型组给予生理盐水灌胃,冬凌草甲素高、中、低剂量组给予75、50、25 mg/(kg·d)冬凌草甲素溶液灌胃。5-FU组给予20 mg/(kg·d)5-FU腹腔注射。5-FU+冬凌草甲素高、中、低剂量组给予20 mg/(kg·d)5-FU腹腔注射,联合75、50、25 mg/(kg·d)冬凌草甲素溶液灌胃,每日1次,连续5 d。观察各组瘤重及抑瘤率,实时荧光定量PCR法检测各组肿瘤组织中Yes相关蛋白(YAP)、具有PDZ结合域的转录共刺激因子(TAZ)的mRNA表达水平,Western blot法检测各组肿瘤组织中细胞周期蛋白D1(CyclinDl)、结缔组织生长因子(CTGF)、生存素(Survivin)、B淋巴细胞瘤-2(Bcl-2)蛋白表达水平。结果40只裸鼠均成瘤,成瘤率100%。和模型组比较,各组均有明显的抑瘤率[(1.079±0.307)g、(1.240±0.250)g、(1.386±0.329)g、(0.783±0.194)g、(0.382±0.214)g、(0.603±0.204)g、(0.897±0.317)g比(1.852±0.584)g,P<0.05或P<0.01],其中5-FU+冬凌草甲素高剂量组抑瘤率最为明显(P<0.01)。与模型组比较,5-FU组、5-FU+冬凌草甲素高、中、低剂量组的YAP和TAZ mRNA相对表达量均显著降低[YAP:(0.319±0.142)、(0.242±0.120)、(0.231±0.114)、(0.267±0.053)比(1.003±0.093);TAZ:(0.542±0.222)、(0.225±0.114)、(0.416±0.185)、(0.446±0.147)比(1.003±0.096),P<0.05或P<0.01],而5-FU+冬凌草甲素高剂量组的TAZ mRNA相对表达量降低最为明显(P<0.01)。与模型组相比,冬凌草甲素中剂量组、5-FU+冬凌草甲素高、中剂量组的CyclinDl、CTGF、Survivin、Bcl-2蛋白表达量不同程度降低[CyclinDl:(0.333±0.050)、(0.284±0.033)、(0.336±0.053)�Objective To assess effects and explore the underlying molecular events of oridonin reversal of drug-resistant liver cancer cells(BEL-7402/5-FU)in nude mouse xenografts.Methods Forty female BALB/c mice of 4 to 6 weeks of age were subcutaneously inoculated with BEL-7402/5-FU cells to establish tumor cell xenografts and after that,these mice were randomly divided into eight groups(n=5 per group),i.e.,the model,oridonin at high,medium,and low doses,5-FU,and 5-FU+oridonin at a high,medium,or low dose.The model group of mice was given normal saline,while treatment groups of mice received oridonin at 75,50,or 25 mg/kg·d,5-FU at 20 mg/kg·d,or 5-FU at 20 mg/kg·d plus oridonin at 75,50,or 25 mg/kg·d once a week for 5 weeks.Mice were monitored daily and tumor xenografts were measured every three days.At the end of experiments,mice were killed using CO2 and the cervical translocation.Tumor xenografts were resected for the real-time fluorescent quantitative PCR analysis of YAP and TAZ mRNA levels or Western blot analysis of CyclinDl,CTGF,Survivin,and Bcl-2 proteins.Results The model showed 100%tumorigenesis rate(40/40 mice).The treatments suppressed tumor xenograft growth compared to the model group of mice(1.079±0.307,1.240±0.250,1.386±0.329,0.783±0.194,0.382±0.214,0.603±0.204,and 0.897±0.317 vs.1.852±0.584 g,respectively;P<0.05 or P<0.01),while the mice treated with 5-FU+high dose of oridonin had the most evident inhibitory rate(0.382±0.214 vs.1.852±0.584 g;P<0.01).Moreover,compared with those of the model mice,expression of YAP(0.319±0.142,0.242±0.120,0.231±0.114,and 0.267±0.053 vs.1.003±0.093)and TAZ(0.542±0.222,0.225±0.114,0.416±0.185,and 0.446±0.147 vs.1.003±0.096)mRNA was significantly reduced in mice received 5-FU and 5-FU+oridonin at a high,medium,or low dose(P<0.05 or P<0.01).Similarly,oridonin at a medium dose or 5-FU plus oridonin at a high or medium dose also significantly decreased expression of Cyclin Dl(0.333±0.050,0.284±0.033,and 0.336±0.053 vs.0.445±0.087),CTGF(0.089±0.007,0.041

关 键 词：冬凌草素 肝癌 耐药 Hippo信号通路 

分 类 号：R285.5[医药卫生—中药学]