褪黑素调控NF-κB信号通路调节胃癌小鼠Th1/Th2免疫平衡及其机制

Melatonin regulates the NF-κB signaling pathway to regulate Th1/Th2 immune balance in mice with gastric cancer and its mechanism

目的探究褪黑素调控NF-κB信号通路调节胃癌小鼠Th1/Th2免疫平衡的机制。方法将50μg(约含有细胞1×106个)胃癌细胞一次性注射到小鼠左侧前肢皮下,在建模7 d后触摸到瘤体生成提示胃癌移植瘤小鼠模型建模成功。10只健康小鼠作为对照组,建模成功的小鼠分为模型组、模型+褪黑素组,每组10只。模型+褪黑素组皮下多点注射褪黑素(10 mg/kg,每天1次,21 d),其余组注射等量的生理盐水。采用游标卡尺、天平测量各组肿瘤体积、重量。通过免疫组化检测肿瘤组织中Ki67和Bcl2评估胃癌细胞增殖和凋亡。通过ELISA检测外周血IFN-γ和IL-4的水平。通过流式细胞术检测外周血Th1和Th2细胞比例。通过qPCR和Western blot检测肿瘤组织和淋巴细胞中NF-κB mRNA和蛋白水平。结果与对照组相比,模型组、模型+褪黑素组的肿瘤体积、质量、Ki67和Bcl2染色强度、IL-4水平、Th2细胞比例、NF-κB mRNA和蛋白表达均显著升高(P<0.05),IFN-γ水平、Th1比例、Th1/Th2比值显著降低(P<0.05);与模型组相比,模型+褪黑素组的肿瘤体积、质量、Ki67和Bcl2染色强度、IL-4水平、Th2细胞比例、NF-κB mRNA和蛋白表达均显著降低(P<0.05),IFN-γ水平、Th1比例、Th1/Th2比值显著升高(P<0.05)。结论褪黑素可能通过调控NF-κB通路抑制胃癌移植瘤小鼠模型的肿瘤生长,并调节Th1/Th2平衡。

Objective To explore the mechanism of melatonin regulating Th1/Th2 immune balance via targeting nuclear factor(NF)-κB signaling pathway in mice with gastric cancer.Methods Gastric cancer cells(50 μg,approximately 1×10~6 cells) were injected subcutaneously into the left forelimb of mice.After 7 days of modeling,tumor formation was felt,indicating that the model of transplanted gastric cancer tumor was successfully established.Ten healthy mice were used as the control group,whereas successfully modeled mice were divided into model and model+melatonin groups(n=10 mice per group).The model+melatonin group was subcutaneously injected daily with melatonin(10 mg/kg,1 time/d,21 d),whereas the other groups were injected with the same amount of normal saline.Tumor volumes and masses were measured using a vernier caliper and balance,respectively.Proliferation and apoptosis of gastric cancer cells in tumor tissues were assessed by immunohistochemical detection of Ki67 and Bcl2.Levels of interferon(IFN)-γ and interleukin(IL)-4 in peripheral blood were detected by ELISA.Ratios of Th1 to Th2 cells in peripheral blood were detected by flow cytometry.Levels of NF-κB mRNA and protein were detected by qPCR and Western blot,respectively,in tumor tissues and lymphocytes.Results Contrast with the control group,the tumor volumes and masses,Ki67 and Bcl2 staining intensities,IL-4 levels,percentage of Th2 cells,NF-κB mRNA and protein expression levels were significantly increased(P<0.05),and the IFN-γ levels,the percentage of Th1 cells and Th1/Th2 ratio were significantly decreased in model and model+melatonin groups(P<0.05).Contrast with model group,tumor volumes and masses,Ki67 and Bcl2 staining intensities,IL-4 levels,percentage of Th2 cells,NF-κB mRNA and protein expression levels were significantly decreased(P<0.05) and the IFN-γ levels,the percentage of Th1 cells and Th1/Th2 ratio were significantly increased in the model+melatonin group(P<0.05).Conclusions Melatonin may inhibit tumor growth in mouse models of gastric cancer and regulate the balance of Th1/Th2 by regulating the NF-κB pathway.