目的:探讨黄芪甲苷Ⅳ(astragaloside Ⅳ,As-Ⅳ)干预低糖介导的肿瘤免疫抑制微环境作用及其分子机制研究.方法:采用MTT实验检测As-Ⅳ在体外低糖微环境下对CD4+T细胞增殖率;采用ELISA实验和qPCR实验检测白细胞介素-2(IL-2)、干扰素-γ(IFN-γ)、CD40L和转化生长因子-β1(TGF-β1)的水平;采用 Western blot 法检测 CD4+T细胞葡萄糖转运蛋白(Glut-1)、糖酵解关键酶[糖激酶(HK)、磷酸果糖激酶1(PFK1)、丙酮酸激酶(PK)]、AKT/mTOR信号和AKT/GSK3β信号通路活性的表达;采用分子对接和加入AKT抑制剂实验进行验证;采用B16-PKM2-OE建立低糖肿瘤微环境动物模型进行验证.结果:MTT结果显示,As-Ⅳ能够促进低糖微环境下CD4+T细胞的增殖(P＜0.05);ELISA实验和qPCR实验检测结果显示,As-Ⅳ可以提高肿瘤组织中IL-2、IFN-γ、CD40L水平,降低TGF-β1 的水平(P＜0.05);Western blot 法检测结果显示,As-Ⅳ能够促进CD4+T细胞细胞膜表面Glut-1蛋白表达,同时呈浓度依赖性上调糖酵解关键酶表达,激活AKT/mTOR信号和AKT/GSK-3β信号;分子对接技术和加入AKT抑制剂实验结果提示As-Ⅳ激活AKT/mTOR信号和AKT/GSK-3β信号;动物实验结果显示As-Ⅳ通过激活低糖微环境下CD4+T细胞增殖与活化发挥抗肿瘤作用.结论:As-Ⅳ通过激活AKT/Glut信号促进低糖微环境下CD4+T细胞增殖与活化发挥抗肿瘤作用.
Abstract
AIM:To investigate the effect of As-tragaloside Ⅳ(As-Ⅳ)on low-glucose mediated tu-mor immunosuppression microenvironment and its molecular mechanism.METHODS:MTT assay was used to detect the effect of As-Ⅳ on the prolifera-tion of CD4+T cells in low-glucose microenviron-ment in vitro.By ELISA experiment and qPCR detec-tion of interleukin 2(IL-2),interferon-gamma(IFN-γ),CD40L and transforming growth factor beta 1(TGF-β1)level;Western blot was used to detect the expression of glucose transporter 1(Glut-1),key glycolytic enzymes(HK,PFK1 and PK),AKT/mTOR signaling pathway and AKT/GSK3β signaling pathway in CD4+T cells.Molecular docking and AKT inhibitor experiments were used to verify the re-sults.B16-PKM2-OE was used to establish a low-glucose tumor microenvironment animal model for verification.RESULTS:MTT assay showed that As-Ⅳ promoted the proliferation of CD4+T cells in low-glucose microenvironment(P＜0.05).The results of ELISA and qPCR experiments showed that As-Ⅳcould increase the levels of IL-2,IFN-γ and CD40L,and reduce the level of TGF-β1 in tumor tissues(P＜0.05).Western blot results showed that As-Ⅳ pro-moted Glut-1 protein expression on the surface of CD4+T cells,up-regulated the expression of glycoly-sis key enzymes,and activated AKT/mTOR and AKT/GSK-3β signaling in a concentration-dependent manner.Molecular docking and join AKT inhibitors As the experiment results indicate-Ⅳ activated AKT/mTOR signaling and AKT/GSK-3β signal;Animal experiments showed that As-Ⅳ exerted anti-tumor effect by activating the proliferation and activation of CD4+T cells in low-glucose microenvironment.CONCLUSION:As-Ⅳ promote sugar by activation of AKT/Glut signal micro environment of CD4+T cell proliferation and activation play a role of anti-tu-mor.
维普
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