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

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20 μg·L^-1IL-4与20 μg·L^-1IL-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。

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

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 20 μg·L^-1 IL-4 and 20 μg·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·L^-1 concentration.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 the survival rate of SW480 cells,increased the apoptosis level of SW480 cells,and up-regulated the expression of apoptotic proteins Bax and Cleaved Caspase-3(P<0.05).CONCLUSION Anemoside A3 can reduce the inhibitory effect of M2 macrophages on apoptosis of SW480 cells,thus improving the chemosensitivity of SW480 cells to 5-FU chemotherapy.The mechanism may be related to regulating STAT6 signal pathway to inhibit M2 polarization of macrophages.