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

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-αmRNA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。

Objective:To investigate the therapeutic effect of Ganmai Dazao Tang on breast cancerrelated 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 highdose(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 depressionlike 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 MAPKmediated NF-κB signaling pathway to reduce neuroinflammation in the treatment of cancer-related depression.