蟾毒灵通过抑制乏氧状态下的乳酸生成调节M2型巨噬细胞极化逆转结肠癌耐药  

Bufalin reverses colon cancer drug resistance by inhibiting lactate production under hypoxia and regulating M2 macrophage polarization

作　　者：贾琳琳 汪红平[1] 池华博文 唐东豪 曹亚琴 吴文韬 李炜[1] 殷佩浩[1] 王啟巍 陈进宝 JIA Lin-lin;WANG Hong-ping;CHI Hua-bo-wen;TANG Dong-hao;CAO Ya-qin;WU Wen-tao;LI Wei;YIN Pei-hao;WANGQi-wei;CHEN Jin-bao(Department ofGeneral Surgery,Shanghai Putuo District Central Hospital,Shanghai 200062,China)

机构地区：[1]上海市普陀区中心医院普外科,上海200062

出　　处：《中国临床药理学杂志》2023年第17期2492-2496,共5页The Chinese Journal of Clinical Pharmacology

基　　金：国家自然科学基金面上基金资助项目(81873137);上海市启明星基金资助项目(扬帆专项)(22YF1441400);上海市普陀区中心医院科研项目基金资助项目(2020366A)。

摘　　要：目的探讨蟾毒灵(Bu)抑制乏氧状态下乳酸生成调节M2型巨噬细胞极化逆转结肠癌耐药的作用。方法(1)将HCT116细胞分为常氧组(HCT116N)、乏氧组(HCT116_(H))和乏氧+BU组(HCT116_(H)+BU),常氧组给予RPMI1640培养基进行培养,乏氧组给予含200μmol·L^(-1)CoCl2的RPMI1640培养基进行培养,乏氧+BU组给予含25 nmol·L^(-1)BU的RPMI1640培养基(含200μmol·L^(-1)CoCl2)进行培养。(2)用佛波酯(200 ng·mL^(-1))将THP^(-1)诱导48 h后成为M0巨噬细胞;将M0巨噬细胞分为空白组(M0)、常氧对照组(HCT116N-M_(φ))、乏氧模型组(HCT116_(H)-M_(φ))、乏氧+乳酸抑制剂(Lactate inhibitor)实验组(HCT116_(H)+Lactate inhibitor-M_(φ))、乏氧+BU实验组(HCT116_(H)+BU-M_(φ)),空白组不作处理,常氧对照组给予HCT116N条件培养基培养96 h,乏氧模型组给予HCT116_(H)条件培养基培养96 h,乏氧+乳酸抑制剂实验组给予HCT116_(H)条件培养基+10μmol·L^(-1)Lactate inhibitor培养96 h,乏氧+BU实验组给予HCT116_(H)+BU细胞条件培养基培养96 h。HCT116细胞分别用1640培养基及前述各组共培养后的细胞条件培养基配制的不同浓度梯度(0、12.5、25、50和100μmol·L^(-1))奥沙利铂(OXA)培养48 h。通过流式细胞术、实时荧光聚合酶链反应、酶联免疫吸附法等实验观察M2型巨噬细胞极化状态,细胞计数试剂盒-8法观察极化后的巨噬细胞对耐药的影响。结果常氧对照组、乏氧模型组的CD11b+CD206+比例分别为(26.83±4.14)%和(38.70±3.40)%,白细胞介素^(-1)0(IL^(-1)0)表达量分别为(108.00±9.17)和(188.33±8.50)pg·mL^(-1),转化生长因子β(TGF-β)表达量分别为(180.67±10.07)和(261.67±10.41)pg·mL^(-1),并且乏氧诱导M2型巨噬细胞极化后促进结肠癌细胞耐药;乏氧+乳酸抑制剂实验组和乏氧模型组IL10表达量分别为(100.33±8.62)和(196.00±8.54)pg·mL^(-1),TGF-β表达量分别为(137.33±10.26)和(224.00±6.56)pg·mL^(-1),并且乳酸诱导M2型巨噬细胞极化后对OXA的敏感性Objective To investigate the effect of bufalin on inhibiting lactic acid production and regulating M2 macrophage polarization in hypoxia to reverse drug resistance in colon cancer.Methods@HCT116 cells were divided into normoxia group(HCT116n),hypoxia group(HCT116)and hypoxia+BU group(HCT116+BU).The normoxia group was cultured with RPMI1640 medium,the hypoxia group was cultured with RPMI1640 medium containing 200μmol·L^(-1)CoCl_(2),and the hypoxia+BU group was cultured with RPMI1640 medium containing 25 nmol·L-'BU(containing 200μmol·L-'CoCl,).②THP-1 was induced to MO macrophages by phorbol ester(200 ng·mL^(-1))for 48 h.MO macrophages were divided into blank group(MO),normoxia control group(HCT116n-M_(φ)),hypoxia model group(HCT116n-M),hypoxia+Lactate inhibitor experimental group(HCT116n+Lactate inhibitor-Mβ),and hypoxia+BU experimental group(HCT116+BU-Mβ).The MO group was not treated.The HCT116n-Mo group was cultured with HCT116n conditioned medium for 96 h,and the HCT116n-Mβwas cultured with HCT116 conditioned medium for 96 h.The HCT116n+Lactate inhibitor-M group was given HCT116_(H)conditioned medium+10μmol·L^(-1)Lactate inhibitor for 96 h,and the HCT116+BU-Mp group was given HCT116+BU cell conditioned medium for 96 h.HCT116 cells were cultured with different concentrations of oxaliplatin(0,12.5,25,50 and 100μmol·L^(-1))for 48 h with 1640 medium and the cell conditioned medium after co-culture in each group.The polarization state of M2 macrophages was observed by flow cytometry,fluorescence polymerase chain reaction and enzyme-linked immunosorbent assay.The effect of polarized macrophages on drug resistance was observed by cell counting kit-8 method.Results The proportion of CD11b^(+)CD206^(+)in the HCT116-Mp group and the HCT116h-Mop group were(26.83±4.14)%and(38.70±3.40)%,respectively.The expression levels of interleukin-10(IL^(-1)0)were(108.00±9.17)and(188.33±8.50)pg·mL^(-1),and the expression levels of transforming growth factor-β(TGF-β)were(180.67±10.07)and(261.67±10.41)pg·mL-I,res

关 键 词：蟾毒灵 乏氧 结肠癌 M2型巨噬细胞 耐药 乳酸 

分 类 号：R28[医药卫生—中药学]