疏肝健脾方对病证结合的乳腺癌模型髓系抑制性细胞-NKT细胞免疫重塑的调控研究

Anxiety and depression of psychological stress are the main causative factors of tumorigenesis and metastasis, which have a severe influence on the life quality and life span of cancer patients. Recent studies have shown that immune suppression induced by Myeloid-derived Suppression Cell (MDSC) plays an important role in the process, and the key mechanisms are probably related with CD8+ T cell apoptosis and NKT differentiation mediated by JAK/ STATs- NO/ROS signal transduction pathway. In traditional Chinese medicine, depression and anxiety belong to liver qi depression, and the pattern of liver depression and spleen deficiency is most commonly seen in the clinic. Our prior study indicated that LSSFF could reverse CD8+ T cell apoptosis through inhibiting the amplification of MDSC in the disease-pattern mouse 4T1 breast cancer model. On basis of this, we design this study by using flow cytometry, in vivo imaging technology in Small Animal, real-time PCR and CFSE assay to dynamically observe the influence of anxiety and depression on breast cancer animal model and the intervention of LSSFF, and further investigate the effect and mechanism of Chinese medicine on the reversing immune escape in breast cancer body from a new viewpoint: whether it is through regulating the immune remodeling of MDSC and NKT mediated by JAK/ STATs- NO/ROS signal transduction pathway? The study will provide scientific evidence for the application of liver-soothing and spleen-fortifying principle in cancer prevention and treatment guided by emotion-thought theory of TCM.

抑郁焦虑负性情绪障碍是促进肿瘤发生、发展的重要因素，但既往缺乏关注和机制探讨。近期研究显示：髓系抑制性细胞（MDSC）所诱导的免疫抑制与其作用机制密切相关，而JAK/ STATs-NO/ROS信号传导途径介导的CD8+T细胞凋亡和NKT细胞促分化作用是MDSC诱导免疫抑制的关键。抑郁焦虑属于中医学"肝郁"范畴，肝郁脾虚是最常见的临床证型，前期研究显示：疏肝健脾方可以通过抑制病证结合的肝郁脾虚小鼠乳腺癌4T1模型体内MDSC的扩增作用，从而逆转CD8+T细胞凋亡。本课题在前期基础上，利用体内及体外共培养研究平台，运用流式细胞术、小动物活体成像、CFSE法等技术，动态观察抑郁焦虑负性情绪障碍对乳腺癌发生、发展的影响及疏肝健脾方的干预作用，从调控JAK/STATs -NO/ROS信号途径介导的MDSC-NKT细胞免疫重塑的新视角，探讨中药逆转乳腺癌免疫逃逸的作用及分子机制,丰富中医情志学说.

抑郁焦虑促进肿瘤发生、发展的重要机制在于诱导机体免疫，本课题首先证实在抑郁荷瘤状态能下调大脑5-HT等神经递质表达，并且分泌大量炎性因子，高表达JAK-STAT信号通路中相关基因，从而激活MDSC扩增，启动免疫抑制。疏肝健脾方相关作用机制从体内、体外两个方面进行了研究。动物实验，建立瘤前肝郁脾虚模型、瘤后肝郁脾虚模型和持续肝郁脾虚模型。抑郁造模后，模型组小鼠与对照组相比，蔗糖水饮用量减少，旷场实验探索能力下降，脑内5-HT等神经递质也相应减少，证明造模成功；BALB/c小鼠接种4T1乳腺癌细胞后，伴随瘤体增大，脾脏MDSC出现了明显聚集和活化并伴脾肿大，抑郁荷瘤小鼠外周血血清中IL-6、IL-4、IL-13、IFN-γ、TGF-β、VEGF水平明显增高，IL-12水平明显下降，脾脏中的CD4+T和CD8+T在抑郁荷瘤模型组早期凋亡最高，增殖能力明显下降。脾脏中分选MDSC后RT-PCR检测相关基因，Arg1、iNOS、JAK1、JAK2、STAT1、STAT2、STAT6表达在抑郁荷瘤模型组均明显上升，免疫组化法检测荷瘤小鼠脾脏ONOO-，硝酸还原法检测脾脏NO和iNOS，NO、iNOS、ONOO-在抑郁荷瘤小鼠组均明显上升，说明肿瘤相关性抑郁所致的免疫抑制涉及JAK/STATs-NO/ROS信号传导通路各个环节。疏肝健脾方具有一定抑制MDSC扩增及功能作用，以中西医结合组作用最为显著；疏肝健脾方联合化疗药物吉西他滨对肿瘤抑郁荷瘤小鼠模型脾脏增大有明显的抑制作用，并且可以降低MDSC比例，同时对抑郁荷瘤小鼠的免疫抑制状态也有明显改善，其主要作用机制在于能够通过下调Arg-1、iNOS、JAK1、JAK2、STAT1、STAT6表达，下调免疫负向调节因子IL-6、IL-4、IL-13、IFN-γ、TGF-β和VEGF表达，上调免疫正向调节因子IL-12表达，减少NO、iNOS、ONOO-生成，抑制MDSC扩增，从而促进T细胞增殖，降低CD8+T细胞凋亡；另一方面，疏肝健脾方联合吉西他滨还能上调iNKT表达并重塑其免疫功能，上调MDSC中CD86、MHCⅡ、CD11c表达，使MDSC趋向成熟，免疫抑制能力减弱，改善荷瘤机体免疫抑制状态。本课题从疏肝健脾、扶正培本的角度，为中医学防治乳腺癌发生发展提供了新的科学依据。