猫爪草总皂苷通过下调信号素4D表达抑制人非小细胞肺癌A549细胞增殖  

Total saponin of Ranunculus ternatus inhibits proliferation of human non-small cell lung cancer A549 cells by down-regulating semaphorin 4D

作　　者：童晔玲[1] 任泽明[1] 陈璇[1] 陈思思 朱佳依 戴关海[1] TONG Ye-ling;REN Ze-ming;CHEN Xuan;CHEN Si-si;ZHU Jia-yi;DAI Guan-hai(Zhejiang Academy of Traditional Chinese Medicine,Key Laboratory of Research and Development of Traditional Chinese Medicine of Zhejiang Province,Hangzhou 310007,China)

机构地区：[1]浙江省中医药研究院,浙江省中药新药研发重点实验室,浙江杭州310007

出　　处：《中国药理学与毒理学杂志》2020年第9期670-676,共7页Chinese Journal of Pharmacology and Toxicology

基　　金：浙江省自然科学基金(LQ18H280010)。

摘　　要：目的探讨猫爪草总皂苷(TSRT)对人非小细胞肺癌A549细胞增殖的影响,并初步探讨其作用机制。方法构建信号素4D(Sema4D)过表达慢病毒载体和阴性对照载体,以感染复数(MOI)10感染A549细胞,用嘌呤霉素1.0 mg·L^-1筛选得到稳定转染细胞株。设感染Sema4D过表达慢病毒的A549细胞为过表达(LV-Sema4D)组,感染Sema4D过表达慢病毒的A549细胞加入TSRT 100 mg·L^-1干预为给药(LV-Sema4D+TSRT)组,感染阴性对照病毒的A549细胞为阴性对照组(LV-NC组),未感染慢病毒的A549细胞为细胞对照组。实时细胞分析系统连续记录96 h细胞指数(CI),荧光定量PCR检测48 h后Sema4D、丛状蛋白B1(PlxnB1)和酪氨酸蛋白激酶(c-Met)mRNA表达;Western印迹法检测Sema4D,PlxnB1和c-Met蛋白表达;免疫荧光法检测Sema4D在细胞中的定位及表达水平。结果LV-NC组CI值与细胞对照组相比无显著差异;与LV-NC组相比,LV-Sema4D组72 h CI值升高(P<0.01);TSRT 100 mg·L^-1干预后,与LV-Sema4D组相比,48,72和96 h CI值均显著降低(P<0.05,P<0.01)。LV-NC组Sema4D,PlxnB1和c-Met mRNA和蛋白表达与细胞对照组相比无显著差异;与LV-NC组相比,LV-Sema4D组上述mRNA和蛋白表达显著升高(P<0.05,P<0.01);TSRT 100 mg·L^-1干预后,与LV-Sema4D组相比,上述mRNA和蛋白表达均显著降低(P<0.05,P<0.01)。LV-NC组Sema4D荧光强度与细胞对照组相比无显著差异,LV-Sema4D组与LV-NC组相比Sema4D荧光强度显著增强(P<0.01);TSRT干预后,与LV-Sema4D组相比,Sema4D荧光强度减弱(P<0.05)。结论TSRT可通过下调Sema4D表达、减少Sema4D与PlxnB1结合、抑制c-Met信号途径,从而抑制A549细胞增殖。OBJECTIVE To explore the effect of total saponin of Ranunculus ternatus(TSRT)on the proliferation of human non-small cell lung cancer A549 cells infected with lentivirus overexpression of semaphorin 4 D(Sema4D)and to investigate the mechanism.METHODS A Sema4D overexpressing lentivirus vector and a negative control vector were constructed.A549 cells were infected with multiplicity of infection(MOI)10,and purine mycin 1.0 mg·L^-1 was screened to obtain stable expressing cell lines.A549 cells infected with Sema4D overexpressing lentivirus were set as the overexpressing(LV-Sema4D)group.A549 cel s infected with Sema4D overexpressing lentivirus and added with TSRT 100 mg·L^-1 were set as the intervention(LV-Sema4D+TSRT)group.A549 cells infected with a negative control virus were set as the negative control group(LV-NC group),and A549 cells that were not infected with lentivirus were set as the cell control group.The real-time cell analysis system continuously recorded the 96 h cell index(CI).After 48 h,the mRNA expressions of Sema4D,PlexinB1(PlxnB1)and CENP-meta(c-Met)were detected by fluorescence quantitative PCR.Western blotting was used to detect protein expressions of Sema4D,PlxnB1 and c-Met.Immunofluorescence method was used to detect the localization and expreesions of Sema4D in cells.RESULTS The CI value of the LV-NC group was not significantly different from that of the cell control group.Compared with the LV-NC group,the CI value of the LV-Sema4D group was obviously increased(P<0.01).Compared with the LV-Sema4D group,the CI values at 48,72 and 96 h in the LV-Sema4D+TSRT group were obviously reduced(P<0.05,P<0.01).The mRNA and protein expressions of Sema4D,PlxnB1 and c-Met in the LV-NC group were not significantly different from those of the cell control group.Compared with the LV-NC group,these mRNA and protein expressions in the LV-Sema4D group were significantly increased(P<0.05,P<0.01).Compared with the LV-Sema4D group,these mRNA and protein expressions of the LV-Sema4D+TSRT group were obviously reduced(P

关 键 词：猫爪草总皂苷 人非小细胞肺癌 信号素4D 细胞增殖 

分 类 号：R285.5[医药卫生—中药学]