基于TLR4/MyD88/NF-κB信号通路探讨黄芪多糖对肺癌小鼠免疫功能的影响及对Th1/Th2的调节作用

Effect of astragalus polysaccharides based on TLR4/MyD88/NF-κB signaling pathway on immune function of lung cancer mice and its regulatory effect on Th1/Th2

目的:探讨黄芪多糖(APS)对肺癌小鼠免疫功能的影响及对Th1/Th2的调节作用及机制。方法:选取C57BL/6J野生型(TLR4^(+/+))和TLR4基因敲除(TLR4^(-/-))小鼠各50只,各随机分为5组:模型组、顺铂组(3 mg/kg)、APS高、中、低剂量组(400、200、100 mg/kg),每组10只,皮下注射Lewis肺癌细胞建立小鼠肺癌移植模型。实验结束后测定小鼠体重、肿瘤组织、胸腺组织和脾组织重量,计算各组小鼠肿瘤抑制率、胸腺指数和脾指数。HE染色观察小鼠肿瘤组织病理变化;流式细胞术检测脾淋巴细胞亚群水平。ELISA试剂盒检测脾组织IL-2、IFN-γ、IL-4和IL-10含量。Western blot和qRT-PCR检测肿瘤组织TLR4/MyD88/NF-κB信号通路相关蛋白和mRNA表达水平。结果:与模型组相比,APS以剂量依赖性抑制Lewis肺癌移植瘤生长,提高肺癌小鼠胸腺指数和脾指数,促进肿瘤细胞凋亡,减少血管生成,提高脾组织CD3^(+)T细胞、CD4^(+)T细胞、CD8^(+)T细胞比例和CD4^(+)/CD8^(+)比值,逆转和平衡Th1/Th2漂移,提高肺癌小鼠免疫功能。APS剂量依赖性降低TLR4^(+/+)组小鼠TLR4、MyD88和NF-κB p65蛋白和mRNA表达,增加Th1型细胞因子IL-2和IFN-γ含量,降低Th2型细胞因子IL-4和IL-10含量(P<0.05);与模型组相比,APS高、中、低剂量组TLR4^(-/-)小鼠TLR4、MyD88和NF-κB p65蛋白和mRNA表达及Th1/Th2细胞因子含量差异无统计学意义(P>0.05)。结论:APS具有抗Lewis肺癌作用和免疫调节作用,其机制可能与抑制TLR4/MyD88/NF-κB信号通路活化有关。

Objective:To investigate effect of astragalus polysaccharide(APS)on immune function of lung cancer mice and its regulatory effect on Th1/Th2 and its mechanism.Methods:50 C57BL/6J wild-type(TLR4^(+/+))and 50 TLR4 gene knockout(TLR4^(-/-))mice were selected,and randomly divided into 5 groups:model group,cisplatin group(3 mg/kg),APS high,medium and low dose groups(400,200,100 mg/kg),10 in each group.Lewis lung cancer cells were injected subcutaneously to establish mice lung cancer transplantation model.After experiment,body weight,tumor tissue,thymus tissue and spleen tissue weight of mice were measured,and tumor inhibition rate,thymus index and spleen index of each group were calculated.HE staining was used to ob⁃serve pathological changes of tumor tissue in mice.Flow cytometry was used to detect splenic lymphocytes subgroup level.ELISA kits were used to detect contents of IL-2,IFN-γ,IL-4 and IL-10 in spleen tissues.Western blot and qRT-PCR were used to detect TLR4/MyD88/NF-κB signaling pathway-related proteins and mRNA expression levels in tumor tissues.Results:Compared with model group,APS dose-dependently inhibited growth of Lewis lung cancer transplanted tumors,improved thymus index and spleen index of lung cancer mice,promoted tumor cell necrosis,reduced angiogenesis,increased spleen tissue CD3^(+)T cells,CD4^(+)T cells,CD8^(+)T cells and CD4/^(+)CD8^(+)ratio,reversed and balanced Th1/Th2 drift,improved immune function of lung cancer mice.APS dose-depend⁃ently reduced expressions of TLR4,MyD88 and NF-κB p65 proteins and mRNA,increased contents of Th1 cytokines IL-2 and IFN-γ,and decreased contents of Th2 cytokines IL-4 and IL-10 in TLR4^(+/+)mice(P<0.05).Compared with model group,there were no sta⁃tistically significant difference in expressions of TLR4,MyD88 and NF-κB p65 proteins and mRNA,and contents of Th1/Th2 cyto⁃kines in APS groups of different concentrations of TLR4^(-/-)mice(P>0.05).Conclusion:APS had anti-lung cancer and immunomodulatory effects,whose mechanism may be related to inhibition of TLR4/MyD88/NF-κB signaling pathway activation.