胃肠安方含药血清诱导胃癌MKN-45细胞铁死亡及其机制  

Weichang'an Prescription-containing Serum Induces Ferroptosis of Gastric Cancer MKN-45 Cells

作　　者：李鑫[1,2] 杨金祖[1] 钱建新 陶丽[1] 陈舲[3] LI Xin;YANG Jinzu;QIAN Jianxin;TAO Li;CHEN Ling(Longhua Hospital,Shanghai University of Traditional Chinese Medicine(TCM),Shanghai 200032,China;Jiangxi Hospital Affiliated Longhua Hospital,Shanghai University of TCM,Nanchang 330006,China;Yueyang Hospital of Integrated Traditional Chinese and Western Medicine,Shanghai University of TCM,Shanghai 200437,China)

机构地区：[1]上海中医药大学附属龙华医院,上海200032 [2]上海中医药大学附属龙华医院江西医院,南昌330006 [3]上海中医药大学附属岳阳中西医结合医院,上海200437

出　　处：《中国实验方剂学杂志》2025年第4期132-139,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(82174450);上海市自然科学基金项目(21ZR1463600)。

摘　　要：目的:观察胃肠安方含药血清对人胃癌细胞铁死亡的影响,并探讨其潜在作用机制。方法:分别灌胃SD大鼠18、36、72 g·kg^(-1)·d^(-1)胃肠安方药液后制备不同剂量的含药血清。不同剂量的胃肠安方含药血清处理MKN-45细胞后,细胞增殖与活性检测法(CCK-8)检测细胞增殖活力的改变;同时添加凋亡抑制剂、坏死性凋亡抑制剂及铁死亡抑制剂观察细胞在胃肠安方含药血清处理后的存活情况;活性氧荧光探针(DCF-DA)检测细胞内活性氧(ROS)水平;脂质过氧化荧光探针(C11-BODIPY)检测细胞内脂质过氧化水平;酶联免疫吸附测定法(ELISA)检测亚铁离子(Fe^(2+))、谷胱甘肽(GSH)和丙二醛(MDA)水平;实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白免疫印迹法(Western blot)分别检测核因子E2相关因子2(Nrf2)、醛酮还原酶1成员B1(AKR1B1)、谷胱甘肽过氧化物酶4(GPX4)、酰基辅酶A合成酶长链家族成员4(ACSL4)、信号转导和转录激活因子3(STAT3)、丝裂原活化蛋白激酶(MAPK)相关分子表达。结果:与空白组比较,胃肠安方含药血清处理后MKN-45细胞活力明显降低(P<0.05,P<0.01),具有时间及浓度依赖性。与胃肠安方组比较,凋亡抑制剂+胃肠安方组及铁死亡抑制剂+胃肠安方组细胞活力明显升高(P<0.05,P<0.01)。与空白组比较,胃肠安方组细胞内ROS、脂质过氧化水平、细胞内Fe^(2+)浓度、MDA含量明显升高(P<0.05,P<0.01),GSH含量明显降低(P<0.05,P<0.01),呈浓度依赖性。与空白组比较,胃肠安方组Nrf2、AKR1B1、GPX4 m RNA和蛋白表达明显降低(P<0.05,P<0.01),ACSL4的m RNA和蛋白表达明显升高(P<0.05,P<0.01),呈浓度依赖性。与空白组比较,胃肠安方组磷酸化(p)-STAT3、p-ERK蛋白表达明显降低(P<0.05,P<0.01),呈浓度依赖性。结论:胃肠安方含药血清可诱导胃癌铁死亡抑制MKN-45细胞增殖,其机制可能与调控STAT3及MAPK通路相关。Objective:To observe the effect of Weichang'an prescription-containing serum on ferroptosis of human gastric cancer cells and explore the possible mechanism.Methods:SD rats were administrated with 18,36,72 g·kg^(-1)·d^(-1)Weichang'an prescription by gavage for preparation of serum samples containing different doses of Weichang'an prescription,which were then used to treat MKN-45 cells.The cell proliferation was examined by the cell counting kit-8(CCK-8).In addition,inhibitors of apoptosis,necroptosis,and ferroptosis were added,and the survival of the cells treated with the serum samples was observed.The fluorescent probe dichlorodihydrofluorescein diacetate(DCF-DA)and the lipid peroxidation sensor C11-BODIPY were employed to detect the intracellular levels of reactive oxygen species(ROS)and lipid peroxidation,respectively.The levels of ferrous ion(Fe^(2+)),glutathione(GSH),and malondialdehyde(MDA)were detected by enzyme-linked immunosorbent assay(ELISA).Realtime PCR and Western blotting were employed to determine the expression of nuclear factor erythroid 2-related factor 2(Nrf2),aldo-keto reductase family 1 member B1(AKR1B1),glutathione peroxidase 4(GPX4),acyl-Co A synthetase long-chain family member 4(ACSL4),signal transducer and activator of transcription 3(STAT3),and mitogen-activated protein kinase(MAPK).Results:Compared with the blank group,Weichang'an prescription-containing serum decreased the viability of MKN-45 cells(P<0.05,P<0.01)in a time-and dose-dependent manner.Compared with the Weichang'an prescription group,the apoptosis inhibitor+Weichang'an prescription group and the ferroptosis inhibitor+Weichang'an prescription group showed increased cell viability(P<0.05,P<0.01).Compared with the blank group,Weichang'an prescription elevated the levels of ROS,lipid peroxidation,and intracellular Fe^(2+)and MDA(P<0.05,P<0.01)and lowered the level of GSH(P<0.05,P<0.01)in a dosedependent manner.Compared with the blank group,Weichang'an prescription down-regulated the m RNA and protein levels of Nrf2,AKR1B1,an

关 键 词：胃癌 胃肠安方含药血清 铁死亡 醛酮还原酶1成员B1 信号转导和转录激活因子3 丝裂原活化蛋白激酶 

分 类 号：R242[医药卫生—中医临床基础] R573[医药卫生—中医学] R285