基于JAK2/STAT3信号通路探讨荔枝核总黄酮对HepG2增殖、迁移与侵袭的影响  

Effect of Total Flavone of Litchi Semen on Proliferation,Migration,and Invasion of HepG2 Cells Based on JAK2/STAT3 Signaling Pathway

作　　者：黎敏航 马晓聪[1] 唐燕 梁瀞云 罗伟生[1] 黄旭平 LI Minhang;MA Xiaocong;TANG Yan;LIANG Jingyun;LUO Weisheng*;HUANG Xuping(Guangxi University of Chinese Medicine,Nanning 530001,China;Guangxi Key Laboratory of Molecular Biology of Preventive Medicine of Traditional Chinese Medicine,Nanning 530001,China;Ruikang Hospital Affiliated to Guangxi University of Chinese Medicine,Nanning 530001,China)

机构地区：[1]广西中医药大学,南宁530001 [2]广西中医药防治医学分子生物重点实验室,南宁530001 [3]广西中医药大学附属瑞康医院,南宁530001

出　　处：《中国实验方剂学杂志》2022年第22期85-92,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(82160834);广西一流学科建设项目(2019XK136)。

摘　　要：目的:观察荔枝核总黄酮(TFL)对人肝癌细胞HepG2株增殖、凋亡、迁移与侵袭的影响。方法:噻唑蓝(MTT)比色法检测不同浓度TFL及顺铂对HepG2细胞增殖的影响;TdT介导的dUTP缺口末端标记法(TUNEL)检测低、中、高质量浓度TFL(70、140、210 mg·L^(-1))及顺铂(60 mg·L^(-1))对HepG2细胞凋亡的影响,并选择TFL最优浓度进行后续实验。将细胞分为空白组、TFL组(140 mg·L^(-1))、TFL+XL019组(140 mg·L^(-1)+0.5μmol·L^(-1))、TFL+TPI-1组(140 mg·L^(-1)+1μmol·L^(-1)),进行细胞划痕实验和小室侵袭实验以验证TFL对HepG2迁移和侵袭的影响;使用细胞免疫荧光技术检测TFL对HepG2细胞上皮间充质转化(EMT)标记物表达的影响;使用蛋白免疫印迹法(Westem blot)对TFL干预后细胞内的酪氨酸蛋白激酶2(JAK2)/信号传导及转录激活因子3(STAT3)信号通路上关键蛋白的表达情况进行检测。结果:MTT比色法表明,与空白组比较,在24、48 h时,各质量浓度的TFL与顺铂均能显著抑制HepG2细胞增殖(P<0.01),24、48 h时TFL对HepG2的半抑制浓度(IC50)分别为(136.7±2.40)、(106.8±1.11)mg·L^(-1),24、48 h时顺铂对HepG2的IC50分别为(58.48±2.04)、(5.15±0.56)mg·L^(-1)。TUNEL法发现各质量浓度TFL均可以诱导HepG2细胞凋亡,由此结果确定TFL 140 mg·L^(-1)为最佳质量浓度。细胞划痕实验表明,与空白组比较,TFL组、TFL+XL019组、TFL+TPI-1组均明显抑制HepG2细胞的迁移能力(P<0.05,P<0.01),与TFL组比较,TFL+XL019组的抑制作用明显增强(P<0.05),TFL+TPI-1组的抑制作用显著减弱(P<0.01)。小室侵袭实验表明,与空白组比较,TFL组、TFL+XL019组、TFL+TPI-1组干预都显著抑制了HepG2细胞的侵袭能力(P<0.01),与TFL组比较,TFL+XL019组的抑制作用明显增强(P<0.05),TFL+TPI-1组的抑制作用则显著减弱(P<0.01)。荧光免疫结果表明,TFL的干预上调HepG2细胞上皮-钙黏蛋白(E-cadherin)的表达,同时下调波形蛋白(Vimentin)的表达,这种作用在TFL+XL019组�Objective:To study the effect of total flavone of Litchi Semen(TFL)on proliferation,apoptosis,migration,and invasion of hepatoma cells HepG2.Method:Methyl thiazolyl tetrazolium colorimetric(MTT)assay was used to detect the effect of different-dose TFL and cisplatin on the proliferation of HepG2 cells.TdT-mediated dUTP nick-end labeling(TUNEL)assay was used to detect the effects of low,medium,and high-dose(70,140,210 mg·L^(-1))of TFL and cisplatin(60 mg·L^(-1))on the apoptosis of HepG2 cells,thus selecting the optimal dose of TFL for the follow-up experiment.HepG2 cells were divided into a blank group,a TFL group(140 mg·L^(-1)),a TFL+XL019 group(140 mg·L^(-1) TFL+0.5μmol·L^(-1) XL019),and a TFL+TPI-1 group(140 mg·L^(-1) TFL+1μmol·L^(-1) TPI-1).The effect of TFL on migration and invasion of HepG2 cells were examined by wound healing test and Transwell invasion assay,and the effect of TFL on the expression of epithelial-mesenchymal transition(EMT)marker in HepG2 cells were examined by cell immunofluorescence assay.Western blot was used to detect the expression of key proteins in Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3)signaling pathway after the intervention by TFL.Result:MTT assay showed that the proliferation of HepG2 cells was significantly inhibited by TFL and cisplatin at 24 and 48 h as compared with blank group(P<0.01),and the half maximal inhibitory concentration(IC50)of TFL on HepG2 cells was(136.7±2.40)mg·L^(-1) at 24 h and(106.8±1.11)mg·L^(-1) at 48 h.The IC50 of cisplatin on HepG2 cells was(58.48±2.04)mg·L^(-1) at 24 h and(5.15±0.56)mg·L^(-1) at 48 h.The results of TUNEL assay showed that TFL induced apoptosis of HepG2 cells.The optimal dose of TFL was 140 mg·L^(-1).The results of wound healing test showed that compared with the blank group,the TFL group,TFL+XL019 group,and the TFL+TPI-1 group significantly inhibited the migration of HepG2 cells(P<0.05,P<0.01).As compared with the TFL group,the inhibitory effect of the TFL+XL019 Group was significant

关 键 词：荔枝核总黄酮 酪氨酸蛋白激酶2/信号传导及转录激活因子3(JAK2/STAT3)信号通路 侵袭 迁移 HepG2 细胞上皮间充质转化(EMT) 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学] R2-031R285.5