白头翁皂苷A3通过M2型巨噬细胞提高人结肠癌SW480细胞对5-FU的化疗敏感性  

Effect of anemoside A3 in improving chemosensitivity of human colon cancer SW480 cells to 5-FU through M2 macrophages

作　　者：范泽萍 陈兰英 刘鹏[1,2] 崔亚茹 罗颖颖[1,2] FAN Ze-ping;CHEN Lan-ying;LIU Peng;CUI Ya-ru;LUO Ying-ying(School of Pharmacy,Jiangxi University of Chinese Medicine,Jiangxi Nanchang 330004,China;National Pharmaceutical Engineering Center for Solid Preparation in Chinese Herbal Medicine,Jiangxi Nanchang 330006,China;Key Laboratory for Evaluation on Anti-tumor Effect of Chinese Medicine by Strengthening Body Resistance to Eliminate Pathogenic Factors,Jiangxi Nanchang 330006,China)

机构地区：[1]江西中医药大学药学院,江西南昌330004 [2]中药固体制剂制造技术国家工程研究中心,江西南昌330006 [3]中药药效(扶正祛邪抗肿瘤)评价重点研究室,江西南昌330006

出　　处：《中国医院药学杂志》2022年第18期1868-1875,共8页Chinese Journal of Hospital Pharmacy

基　　金：国家自然科学基金项目(编号:81860720);江西省中医药重点研究室建设项目。

摘　　要：目的:观察白头翁皂苷A3(A3)通过干预巨噬细胞极化增强人结肠癌SW480细胞对5-氟尿嘧啶(5-FU)化疗的敏感性,并探讨其作用机制。方法:采用CCK8法检测A3对人单核THP-1细胞存活率的影响,确定A3的安全作用浓度;采用佛波酯(PMA)诱导THP-1细胞分化为M0型巨噬细胞,将M0型巨噬细胞分为M0对照组,M2模型组,A3低、中、高剂量干预组。其中M2模型组给予20μg·L^(-1)IL-4与20μg·L^(-1)IL-13进行造模,A3干预组在造模的同时给予低、中、高剂量(50,75,100 mg·L^(-1))A3进行干预。采用流式细胞术检测M2型巨噬细胞表面抗原CD206的表达;采用RT-PCR法检测M2型巨噬细胞极化因子CD206、IL-10、CCL22、CCL18 mRNA的表达;采用Western blot法检测细胞内p-STAT6、CD206、IL-10、CCL22蛋白的表达。A3干预巨噬细胞M2型极化后的细胞上清液作为条件培养基用于考察SW480细胞对5-FU的化疗敏感性,采用CCK8法检测SW480细胞的存活率;采用Annexin-FITC/PI双染法及Western blot法检测SW480细胞凋亡水平,以及Bax、Cleaved Caspase-3蛋白的表达。结果:A3在50,75,100 mg·L^(-1)浓度时对THP-1细胞存活率没有显著影响。A3呈剂量依赖性减少CD206阳性细胞表达比例,下调p-STAT6、CD206、IL-10、CCL22、CCL18基因与蛋白的表达(P<0.05,P<0.01)。A3干预巨噬细胞M2型极化后的条件培养基显著提高了SW480细胞对5-FU的敏感性,表现为SW480细胞的存活率下降、凋亡率升高,以及促凋亡蛋白Bax、Cleaved Caspase-3表达上调(P<0.05)。结论:A3能够减弱M2型巨噬细胞对SW480细胞凋亡的抑制作用从而提高SW480细胞对5-FU的化疗敏感性,作用机制可能与A3调控STAT6信号通路抑制巨噬细胞M2型极化有关。OBJECTIVE To observe that anemoside A3(A3)enhances the sensitivity of human colon cancer SW480 cells to 5-fluorouracil(5-FU)chemotherapy by interfering with macrophage polarization,and to explore its mechanism.METHODS CCK8 assay was used to detect the effect of anemoside A3 on the survival rate of human mononuclear THP-1 cells,so as to screen the safe concentration of anemoside A3 on THP-1 cells.Phorbol ester(PMA)was used to induce THP-1 cells to differentiate into M0 macrophages,M0 macrophages were divided into M0 control group,M2 model group and A3 low,medium and high dose intervention groups.The M2 model group was given 20μg·L^(-1)IL-4 and 20μg·L^(-1)IL-13,and the A3 intervention group was given low,medium and high doses(50,75,100 mg·L^(-1))of A3.Flow cytometry was used to detect the expression of M2 macrophage surface maker CD206.RT-PCR was used to detect the mRNA expression of M2 macrophage polarization factors CD206,IL-10,CCL18,and CCL22.Western blot was used to detect the expression of p-STAT6,CD206,IL-10 and CCL22 protein in M2 macrophages.The supernatant after anemoside A3 interfered with M2 polarization of macrophages was used as conditioned medium for the experiment of SW480 cells sensitivity to 5-FU,CCK8 assay was used to detect the survival rate of SW480 cells.Annexin-FITC/PI double staining method was used to detect the apoptosis level of SW480 cells.Western blot assay was used to detect the expression of apoptosis proteins Bax and Cleaved caspase-3 in SW480 cells.RESULTS Anemoside A3 had no significant effect on THP-1 cells viability at 50,75,100 mg·Lconcentration.Anemoside A3 intervention decreased the M2 macrophage marker CD206,reduced the gene and protein expression levels of p-STAT6,CD206,IL-10,CCL22 and CCL18 in a dose-dependent manner(P<0.05,P<0.01).Conditioned medium after anemoside A3 interfered with M2 polarization of macrophages significantly increased the sensitivity of SW480 cells to 5-FU.The results showed that the combination of 5-FU and anemoside A3 conditioned medium reduced t

关 键 词：白头翁皂苷A3 巨噬细胞极化 STAT6 人结肠癌SW480细胞 5-FU 化疗敏感性 

分 类 号：R967[医药卫生—药理学]