基于巨噬细胞极化观察白术内酯Ⅱ对胃癌细胞的作用

Observation on Effect of Atractylodesin Ⅱ on Gastric Cancer Cells Based on Macrophage Polarization

目的:研究白术内酯Ⅱ对巨噬细胞极化的影响并探讨其发挥抗肿瘤的作用机制。方法:用佛波酯(PMA)诱导THP-1细胞分化成巨噬细胞,噻唑蓝(MTT)比色法检测不同浓度白术内酯Ⅱ作用不同时间,对巨噬细胞生长的影响,筛选出白术内酯Ⅱ的安全给药浓度。不同浓度白术内酯Ⅱ作用24 h,巨噬细胞与胃癌细胞共培养,光镜下观察2种细胞的存活状态,MTT比色法检测胃癌细胞的增殖变化,筛选出白术内酯Ⅱ的有效给药浓度。将细胞分为空白组、模型组、白术内酯Ⅱ(200,100,50 mg·L-1)组。划痕实验观察不同浓度白术内酯Ⅱ作用后巨噬细胞对胃癌细胞迁移及形态的影响。流式细胞术(FCM)检测M1,M2型巨噬细胞表面标志CD86,CD206表达;实时荧光定量聚合酶链式反应(Real-time PCR)及蛋白免疫印迹法(Western blot)检测M1,M2型巨噬细胞相关肿瘤坏死因子(TNF)-α,人类白细胞抗原2(HLA-DRA),CD80,转化生长因子-β(TGF-β),白细胞介素(IL)-10和IL-6 mRNA和蛋白表达;Western blot检测巨噬细胞内磷脂酰肌醇激酶(PI3K)和磷酸化(p)-PI3K蛋白表达。结果:当白术内酯Ⅱ质量浓度为1,10,50,100,200 mg·L-1时,对巨噬细胞生长无抑制作用;与模型组比较,50,100,200 mg·L-1白术内酯Ⅱ作用的巨噬细胞可显著抑制胃癌细胞增殖(P<0.01);与模型组比较,白术内酯Ⅱ(200,100 mg·L-1)组胃癌细胞迁移率降低(P<0.05);白术内酯Ⅱ(200,100,50 mg·L-1)组M1型巨噬细胞表面标志CD86表达增高(P<0.05,P<0.01),白术内酯Ⅱ(200 mg·L-1)组M2型巨噬细胞表面标志CD206表达下降(P<0.05);白术内酯Ⅱ(200,100 mg·L-1)组M1型巨噬细胞相关细胞因子TNF-α,HLA-DRA,CD80 mRNA表达增高(P<0.05,P<0.01),白术内酯Ⅱ(200 mg·L-1)组TNF-α蛋白表达增高(P<0.05);白术内酯Ⅱ(50 mg·L-1)组M2型巨噬细胞相关细胞因子TGF-βmRNA表达显著降低(P<0.01),白术内酯Ⅱ(200 mg·L-1)组IL-10,IL-6蛋白表达降低(P<0.05,P<0.01);白术内酯Ⅱ(200,100 mg·L-1)组p-PI3K蛋白表达降低(P<0.05,P<0.01)。结论:白术内酯Ⅱ通过减少巨噬细胞内p-PI3K的表达,诱导巨噬细胞向M1型极化,进而抑制胃癌增殖和迁移。

Objective:To investigate the anti-tumor effect mechanism of atractylenolide Ⅱ by studying its effect on macrophage polarization. Method: Phorbol myristate acetate(PMA)was used to induce THP-1 cells differentiation into macrophages,and methylthiazolyldiphenyl-tetrazolium bromide(MTT)colorimetric assay was used to detect the effect of different concentrations of atractylenolide Ⅱ on macrophage growth at different time points to screen out the safe concentration of atractylenolide Ⅱ. The macrophages were treated with different concentrations of atractylenolide Ⅱ for 24 hours and then were co-cultured with gastric cancer cells. The survival of the two types of cells was observed under light microscope. The proliferation of gastric cancer cells was detected by MTT assay to determine the effective administration concentrations of atractylenolide Ⅱ. Cells were divided into blank group,model group,atractylenolide Ⅱ high dose group(200 mg·L-1),atractylenolide Ⅱ medium dose group(100 mg·L-1),and atractylenolide Ⅱ low dose group(50 mg·L-1). Wound healing assay was carried out to observe the effects of different concentrations of atractylenolide Ⅱ on the migration and morphology of gastric cancer cells. The expression levels of M1 and M2 macrophage surface markers CD86 and CD206 were detected by flow cytometry analysis(FCM). Quantitative polymerase chain reaction(Real-time PCR)and Western blot were used to detect M1,M2 macrophage-associated tumor necrosis factor(TNF)-α,human leukocyte antigen 2(HLA-DRA),CD80,transforming growth factor(TGF)-β,interleukin(IL)-10 and IL-6 genes and protein expression. Western blot was used to detect intracellular phosphatidyl inositol kinase(PI3 K)and p-PI3 K protein expression in macrophages. Result: When the concentration of atractylenolide Ⅱ was 1,10,50,100,200 mg·L-1,it showed no inhibition on macrophage growth. As compared with the model group,macrophages treated with 50,100,200 mg·L-1 atractylenolide Ⅱsignificantly inhibited tumor cell proliferation(P<0.01). As compared with the model group,the migration rate of tumor cells in the atractylenolide Ⅱ(200,100 mg·L-1)groups decreased(P<0.05). The expression levels of CD86 on M1 macrophage surfacen in the atractylenolide Ⅱ(200,100,50 mg·L-1)groups were increased(P<0.05,P<0.01),and the expression levels of CD206 on M2 macrophagen in the atractylenolide Ⅱ(200 mg·L-1)group were decreased(P<0.05). The expression levels of M1 macrophage-associated cytokines TNF-α,HLADRA,CD80 mRNA in the atractylenolide Ⅱ(200,100 mg·L-1)groups were increased(P<0.05,P<0.01),and TNF-α protein expression in the atractylenolide Ⅱ(200 mg·L-1)group was increased(P<0.05),M2 type macrophage-associated cytokine TGF-β mRNA expression levels in the atractylenolide Ⅱ(50 mg·L-1)group were decreased,and IL-10,IL-6 protein expression levels in the atractylenolide Ⅱ(200 mg·L-1)group were decreased(P<0.05,P<0.01). The expression levels of p-PI3 K protein in the atractylenolide Ⅱ (200,100 mg·L-1)groups were also decreased(P<0.05,P<0.01). Conclusion: Atractylenolide Ⅱ could induce the polarization of macrophages to M1 type by reducing the expression of p-PI3 K in macrophages and inhibiting the proliferation and migration of gastric cancer cells.