摘要
目的探讨黄芪-莪术-重楼配伍对巨噬细胞极化的影响及其抑制结直肠癌细胞增殖、迁移的作用机制。方法使用佛波酯(Phorbol 12-myristate 13-acetate,PMA)和白细胞介素-4(Interleukin-4,IL-4)刺激THP-1细胞建立M2型巨噬细胞极化模型。实验分为M0组(PMA处理)、M2组(PMA+IL-4处理)、M2+黄芪-莪术-重楼组(PMA+IL-4+黄芪-莪术-重楼配伍处理)。CCK-8检测黄芪-莪术-重楼配伍冻干粉对巨噬细胞活力影响;qPCR和Western blot检测巨噬细胞极化标志物、谷氨酰胺酶(Glutaminase,GLS)mRNA和蛋白表达;ELISA检测细胞上清白细胞介素-10(Interleukin-10,IL-10)、转化生长因子-β(Transforming growth factor-β,TGF-β)、肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)含量;CCK-8和Transwell实验检测黄芪-莪术-重楼配伍干预巨噬细胞的培养上清,即条件培养基(Conditioned medium,CM),对结直肠癌细胞HCT116增殖和迁移能力影响。结果与M0组相比,M2组IL-10、甘露糖受体(Mannose receptor,CD206)、精氨酸酶1(Arginase 1,ARG1)、GLS mRNA和蛋白表达显著升高(P<0.01,P<0.001),巨噬细胞分泌IL-10、TGF-β水平显著升高(P<0.01,P<0.001);与M2组相比,M2+黄芪-莪术-重楼配伍组IL-10、CD206、ARG1、GLS mRNA和蛋白表达显著降低(P<0.05,P<0.01),TNF-α、诱导型一氧化氮合酶(Inducible nitric oxide synthase,iNOS)mRNA和蛋白表达显著升高(P<0.05,P<0.01,P<0.001),白细胞介素-1β(Interleukin-1β,IL-1β)mRNA表达水平显著升高(P<0.01),细胞上清中IL-10、TGF-β水平显著降低(P<0.05,P<0.01),TNF-α水平显著升高(P<0.01)。CCK-8和Transwell结果显示,与M0-CM组相比,M2-CM组促进HCT116细胞增殖和迁移(P<0.01,P<0.001),M2+黄芪-莪术-重楼-CM组较M2-CM组显著抑制HCT116细胞增殖并使细胞迁移数显著减少(P<0.01,P<0.001)。结论黄芪-莪术-重楼配伍可通过调控巨噬细胞极化,抑制结直肠癌细胞增殖、迁移,其机制可能与谷氨酰胺代谢关键酶GLS表达变化有关。
OBJECTIVE To investigate the effect of Huangqi-Ezhu-Chonglou combination on macrophage polarization and its mechanism of inhibiting colorectal cancer(CRC)cells proliferation and migration.METHODS THP-1 cells were stimulated with phorbol 12-myristate 13-acetate(PMA)and interleukin-4(IL-4)to establish M2 macrophage polarization model.The experiment was divided into M0 group(PMA treatment),M2 group(PMA+IL-4 treatment),and M2+Huangqi-Ezhu-Chonglou combination group(PMA+IL-4+Huangqi-Ezhu-Chonglou combination treatment).The effect of Huangqi-Ezhu-Chonglou combination freeze-dried powder on the viability of macrophage was detected by CCK-8 method.The expression of macrophage polarization markers,glutaminase(GLS)mRNA and protein was detected by qPCR and Western blot.The levels of interleukin-10(IL-10),transforming growth factor-β(TGF-β)and tumor necrosis factor-α(TNF-α)in cell supernatant were detected by ELISA.CCK-8 method and Transwell assays were used to detect the proliferation and migration of HCT116 cells intervened by the supernatant of macrophage culture treated with Huangqi-Ezhu-Chonglou combination,namely conditioned medium(CM).RESULTS Compared with the M0 group,the expression levels of IL-10,mannose receptor(CD206),arginase 1(ARG1),and GLS mRNA and protein in the M2 group were significantly increased(P<0.01,P<0.001),the levels of IL-10 and TGF-βsecreted by macrophages were significantly increased(P<0.01,P<0.001);compared with the M2 group,the M2+Huangqi-Ezhu-Chonglou combination group had significantly reduced IL-10,CD206,ARG1,and GLS mRNA and protein expression(P<0.05,P<0.01),the mRNA and protein levels of TNF-αand inducible nitric oxide synthase(iNOS)were significantly increased(P<0.05,P<0.01,P<0.001),the interleukin-1β(Interleukin-1β,IL-1β)mRNA expression significantly increased(P<0.01),and the contents of IL-10 and TGF-βin the cell supernatant significantly decreased(P<0.05,P<0.01),while TNF-αcontent significantly increased(P<0.01).CCK-8 and Transwell results showed that compared with the M0-CM
作者
杜利莉
王钢
梁研
赵凡
应佳辉
尹刚
唐德才
卞勇
DU Lili;WANG Gang;LIANG Yan;ZHAO Fan;YING Jiahui;YIN Gang;TANG Decai;BIAN Yong(Laboratory Animal Center,Nanjing University of Chinese Medicine,Nanjing 210023,China;School of Chinese Medicine,Nanjing University of Chinese Medicine,Nanjing 210023,China;School of Pharmacy,Nanjing University of Chinese Medicine,Nanjing 210023,China)
出处
《南京中医药大学学报》
CAS
CSCD
北大核心
2024年第2期137-144,共8页
Journal of Nanjing University of Traditional Chinese Medicine
基金
国家自然科学基金面上项目(82274116)。
