基于MAPK/NF-κB信号通路探讨甘麦大枣汤对乳腺癌相关抑郁症的治疗作用及机制

Ganmai Dazao Tang Treats Breast Cancer-related Depression via MAPK/NF-κB Signling Pathway

摘要:

目的 观察甘麦大枣汤对乳腺癌相关抑郁的抗抑郁作用，并从丝裂原活化蛋白激酶（MAPK）/核转录因子-κB（NF-κB）通路探讨其调控免疫炎症及神经递质的作用机制。方法 BALB/c小鼠随机分为正常组、模型组、氟西汀组（氟西汀5 mg·kg^-1·d^-1）、甘麦大枣汤低（20 g·kg^-1）、高（40 g·kg^-1）剂量组，每组10只。建立小鼠乳腺癌4T1原位移植瘤诱导小鼠抑郁样行为模型，通过悬尾实验和强迫游泳实验评价小鼠抑郁样行为； 实时荧光定量聚合酶链式反应 （Real-time PCR）检测大脑皮层中白细胞介素（IL）-17A、叉头框蛋白P3（FoxP3）、IL-1β、IL-6、肿瘤坏死因子-α（TNF-α）mRNA表达；流式细胞术检测脾和胸腺免疫细胞亚群比例；液相色谱-质谱联用技术（HPLC-MS）/MS检测大脑皮层内神经递质含量；蛋白免疫印迹法（Western blot）检测MAPK和NF-κB通路活化。结果 与模型组比较，甘麦大枣汤40 g·kg^-1连续给药4周能明显降低乳腺癌荷瘤动物悬尾和强迫游泳不动时间（P<0.05）；IL-1β、IL-17A、TNF-α mRNA表达水平明显降低（P<0.05）；T细胞、CD4⁺T细胞、B细胞、辅助性T细胞17（Th17）、调节性T细胞（Treg）比例升高，CD8⁺ T细胞比例下降（P<0.05）；5-羟吲哚乙酸（5-HIAA）、犬尿氨酸（Kyn）含量与犬尿氨酸/色氨酸（Kyn/Trp）明显下降（P<0.05），5-羟色胺（5-HT）含量增加；磷酸化细胞外信号调节激酶（p-ERK）、磷酸化p38 MAPK（p-p38 MAPK）、磷酸化NF-κB p65亚基（p-NF-κB p65）蛋白表达水平明显降低（P<0.05）。结论 甘麦大枣汤能够有效治疗肿瘤相关抑郁，其治疗机制与抑制大脑皮层p38 MAPK和ERK，MAPK途径介导的NF-κB信号通路的激活，抑制脑内IL-1β、IL-17A、TNF-α等炎性因子表达，调节脑内5-HT代谢和Kyn/Trp平衡，增加脑内5-HT含量，改善神经炎症有关。

Abstract:

Objective To investigate the therapeutic effect of Ganmai Dazao Tang on breast cancer-related depression and explore the mechanism of the decoction in regulating immune inflammation and neurotransmitters via the mitogen-activated protein kinase （MAPK）/nuclear factor-κB （NF-κB） pathway.Method BALB/c mice were randomized into control， model， fluoxetine （5 mg·kg^-1·d^-1）， and low- and high-dose （crude drug 20 and 40 g·kg^-1， respectively） Ganmai Dazao Tang groups （n=10）. The mouse model of 4T1 orthotopic transplantation-induced breast cancer-related depression-like behavior was established. The depression-like behavior of mice was assessed by the tail suspension test and the forced swimming test. RT-qPCR was employed to determine the mRNA levels of interleukin （IL）-17A， forkhead box P3 （FoxP3），IL-1β， IL-6， and tumor necrosis factor-α （TNF-α） in the cerebral cortex. Flow cytometry was employed to measure the proportions of immune cell subsets in the spleen and thymus. HPLC-MS/MS was employed to measure neurotransmitter levels in the cerebral cortex. Western blotting was employed to detect the activation of the MAPK/NF-κB pathway.Result Compared with the model group， administration of Ganmai Dazao Tang at a dose of 40 g crude drug·kg^-1 continuously for 4 weeks shortened the immobility time of modeled mice in the tail suspension and forced swimming tests （P<0.05）， down-regulated the mRNA levels of IL-1β， IL-17A， and TNF-α （P<0.05）， increased the proportions of T cells， CD4⁺ T cells， B cells， helper T 17 （Th17） cells， and regulatory T （Treg） cells， and reduced the proportion of CD8⁺ T cells （P<0.05）. Furthermore， it lowered the levels of 5-hydroxyindoleacetic acid （5-HIAA） and kynurenine （Kyn）， decreased the kynurenine/tryptophan （Kyn/Trp） ratio （P<0.05）， increased the content of 5-hydroxytryptamine （5-HT）， and down-regulated the protein levels of phosphorylated extracellular signal-regulated kinase （p-ERK）， phosphorylated p38 MAPK， and phosphorylated nuclear factor-κB p65 （P<0.05）.Conclusion Ganmai Dazao Tang can down-regulate the expression of inflammatory cytokines such as IL-1β， IL-17A， and TNF-α， restore 5-HT metabolism and Kyn/Trp balance， increase the 5-HT content， and reduce the activation of p38 MAPK， ERK， and the MAPK-mediated NF-κB signaling pathway to reduce neuroinflammation in the treatment of cancer-related depression.