参桃软肝方抑制人肝癌细胞HepG2活性的作用机制研究  

Study on the Mechanism of Shentao Ruangan Formula Inhibiting Cell Viability of Human Hepatoma Cells HepG2

作　　者：黄海福[1,2] 付艳丽 符必谦 赖思思 Huang Haifu;Fu Yanli;Fu Biqian;Lai Sisi(Shenzhen Hospital of Guangzhou University of Chinese Medicine,Shenzhen 518000,China;Shenzhen Medical Oncology Center of Chinese Medicine,Shenzhen 518000,China)

机构地区：[1]广州中医药大学深圳医院,深圳518000 [2]深圳市中医肿瘤医学中心,深圳518000

出　　处：《世界科学技术-中医药现代化》2023年第7期2353-2364,共12页Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology

基　　金：广东省中医药管理局面上项目:参桃软肝方调控肝癌细胞焦亡机制研究(20211335),负责人:黄海福;深圳市福田区卫生健康局公益性重点项目(FTWS2020015):“参桃软肝丸”干预肝癌高危人群的疗效观察,负责人:黄海福。

摘　　要：目的探讨参桃软肝方抗肝癌的作用及机制。方法四甲基噻唑蓝(Methyl thiazolyl tetrazolium,MTT)法检测参桃软肝方对人肝癌细胞HepG2活性及胞内磷脂酰肌醇激酶/蛋白激酶B/丝裂原活化蛋白激酶(Phosphatidylinositol-3-kinase/Protein Kinase B/mitogen-activated protein kinase,PI3K/Akt/MAPK)抑制剂:LY294002、Atk抑制剂(Akt inhibitor,Akti)、分裂原活化抑制剂(MEK inhibitor,MEKi)、c-Jun氨基末端蛋白激酶抑制剂(c-Jun N-terminal protein kainse inhibitor,JNKi)、凋亡抑制剂(Z-VAD)、自噬抑制剂(Wortmanin)、铁死亡抑制剂(Ferrostatin-1)、细胞坏死抑制剂(Necrostatin-1)对参桃软肝方抑制人肝癌细胞HepG2活性的影响;乳酸脱氢酶(Lactate dehydrogenase,LDH)法检测参桃软肝方对人肝癌细胞HepG2的毒性作用;实时荧光定量(Real-time PCR)检测参桃软肝方对人肝癌细胞HepG2焦亡标志物白介素1β(Interleukin-1β,IL-1β)的影响;蛋白质印迹法(Western blot)检测参桃软肝方对人肝癌细胞HepG2中Akt/ERK信号通路关键蛋白Akt、ERK、P-ERK蛋白表达的影响,凋亡信号通路关键蛋白半胱氨酸-天冬氨酸蛋白酶(Caspase 3)、聚腺苷二磷酸核糖聚合酶(PARP)、B细胞淋巴瘤/白血病-2基因(Bcl2)、Bcl2-Associated X的蛋白质(Bax),细胞焦亡标志物卵裂半胱天冬酶1(Cleavaged Caspase-1)蛋白表达的影响。结果参桃软肝方及醇提上清和沉淀都能有效抑制人肝癌细胞HepG2的活性(P<0.05),LY294002、Wortmanin、Akti、MEKi、JNKi、Z-VAD、Ferrostatin-1、Necrostatin-1对参桃软肝方、醇提上清和沉淀抑制人肝癌细胞HepG2活性没有显著的影响;参桃软肝方促进人肝癌细胞HepG2中LDH的释放,引起细胞毒性;参桃软肝方抑制人肝癌细胞HepG2焦亡标志物IL-1β和Cleavaged Caspase-1的表达;参桃软肝方降低人肝癌细胞HepG2中Akt的蛋白表达,升高Caspase3蛋白表达量。结论参桃软肝方具有抑制肝癌细胞活性、抗肝癌的作用,其作用机制可能与自噬、坏死�Objective To explore the effects and mechanism of Shentao Ruangan Prescription on hepatocellular carcinoma.Methods MTT assay was used to detect the activity of Shentao Ruangan Prescription against HepG2 cells and the effects of PI3K/Akt/MAPK inhibitors(LY294002,Akti,MEKi,JNKi),apoptosis inhibitor(Z-VAD)and autophagy inhibitor(Wortmanin)in human hepatoma cells.),ferroptosis inhibitor(Ferrostatin-1)and cell necrosis inhibitor(Necrostatin-1)on the inhibition of HepG2 activity of human hepatoma cells by Shentao Ruangan Prescription.LDH method was used to detect the toxicity of Shentao Ruangan Prescription on HepG2 cells;Real-time PCR experiment tested the effect of Shentao Ruangan Prescription on the pyroptosis marker IL-1β in human hepatoma cells HepG2;Western blot experiment explored the effect of Shentao Ruangan Prescription on the Akt/ERK signaling pathway in human hepatoma HepG2 cells and the expression of Akt,ERK,P-ERK,the key proteins of apoptosis signaling pathway Caspase 3,PARP,Bcl2,Bax,and the expression of pyroptosis marker Cleavaged Caspase-1.Results Shentao Ruangan Prescription Can effectively inhibit the activity of human hepatoma HepG2 cells.LY294002,Wortmanin,Akti,MEKi,JNKi,Z-Vad,Ferrostatin-1,Necrostatin-1 had no significant effect on the inhibition of HepG2 activity by total extract(water extract),supernatant and precipitation.Shentao Ruangan Prescription promoted the release of LDH in human hepatoma HepG2 cells,causing cytotoxicity.It inhibited HepG2 pyroptosis markers in human hepatoma cells and the expression of IL-1β and Cleavaged Caspase-1 and decreased the protein expression of Akt in human hepatoma cells HepG2 but inversely increased Caspase3 protein expression.Conclusion Shentao Ruangan Prescription has the effect of inhibiting the activity of hepatocellular carcinoma cells and anti-hepatocellular carcinoma,and its mechanism may be related to inhibiting the expression of Akt and promoting the expression of Caspase3.At the same time,Shentao Ruangan Prescription reduces the expressions of IL

关 键 词：参桃软肝方 肝癌细胞HEPG2 细胞焦亡 细胞凋亡 PI3K/Akt/MAPK信号通路 

分 类 号：R285.5[医药卫生—中药学]