基于细胞焦亡通路探讨疏肝健脾方调控乳腺癌MDSC-NKT细胞免疫编辑机制研究

Cancer-related depression (CRD) has attracted more and more attention in clinic because it plays important role in treatment effect and long-term survival of cancer patients. The latest research shows that CRD may facilitate the formation of tumor inflammatory microenvironment and induce immunosuppression. The key point lies on the death of CD8+ T cell induced by Myeloid–derive suppressor cells (MDSCs) and Natural Killer T （NKT） cell through NLRP3 inflammasome-mediated pyroptosis signaling pathways. In traditional Chinese medicine, CRD belongs to liver qi depression, and Liver-soothing and Spleen- fortifying Formula （LSSFF）is an empirical formula in our department. Previous studies supported by NSFC have shown that LSSFF could reverse CD8+ T cell apoptosis through inhibiting the MDSC amplification and promote the differentiation of MDSC by NKT in the disease-pattern mouse 4T1 breast cancer model. On basis of this, we hypothesized that the core mechanism of LSSFF is to regulate CD8 + T cells pyroptosis mediated by NLRP3 inflammasome, and design this study to dynamically observe the influence of CRD 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 through means of flow cytometry, in vivo imaging technology in Small Animal, high content screening, etc. We hope to figure out furtherly whether LSSFF acts through regulating the immune editing of MDSC and NKT mediated by Tim-3 /Galectin-9- ROS/p62- NLRP3 inflammasome mediated pyroptosis signaling pathways? 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.

肿瘤相关性抑郁（CRD）是影响患者治疗效果及远期生存的重要因素，最新研究显示促进肿瘤炎症微环境形成及诱发免疫抑制是CRD重要作用机制。基于NLRP3炎症小体介导的细胞焦亡通路，髓系抑制细胞（MDSC）-NKT细胞诱导CD8+T细胞死亡可能是关键环节。CRD属于肝郁范畴，疏肝健脾方是临床经验方，前期研究显示：该方具有抑制抑郁所致肝郁脾虚小鼠乳腺癌MDSC扩增，提高NKT细胞促MDSC分化成熟，逆转CD8+T细胞死亡作用。调控NLRP3炎症小体介导的CD8+T细胞焦亡可能是该方核心作用机制。本课题拟利用体内研究和体外共培养方法，动态观察抑郁障碍对术后、荷瘤乳腺癌复发、转移影响及疏肝健脾方的干预作用，从Tim-3/Galectin-9-ROS/p62-NLRP3炎症小体介导的细胞焦亡通路重塑MDSC-NKT细胞免疫编辑过程新视角，进一步揭示抑郁导致肿瘤进展免疫分子机制和中药干预作用。

肿瘤相关性抑郁（CRD）是影响患者治疗效果及远期生存的重要因素，既往研究显示CRD可促进肿瘤炎症微环境形成及诱发免疫抑制。.本课题动态观察不同时段抑郁障碍的荷瘤及术后状态可下调大脑5-HT等神经递质表达，并且分泌大量炎性因子，促进负向免疫细胞MDSC、TAM扩增，抑制正向免疫细胞NKT、CD4+T、CD8+T。疏肝健脾方相关作用及机制从体内、体外两个方面进行了研究。动物实验，建立瘤前抑郁障碍模型、瘤后抑郁障碍模型、持续抑郁障碍模型、瘤前抑郁障碍乳腺癌术后模型、持续抑郁障碍乳腺癌术后模型。小鼠蔗糖水饮用量减少、旷场实验探索能力下降、脑内5-HT水平降低，证明抑郁障碍造模成功，经药物干预后相应指标改善；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，经药物干预后瘤体及脾脏缩小；抑郁荷瘤小鼠外周血血清中IL-6、IL-1β、IL-18、IL-17、TNF-α等细胞因子表达水平明显增高，脾脏NKT、CD4+T和CD8+T在抑郁荷瘤模型组水平下降，脾脏TAM、MDSC水平提高，经药物干预后细胞因子水平降低、正向免疫细胞比例提高、负向免疫细胞比例降低，说明抑郁可以促进乳腺癌发生发展及复发转移，并促进荷瘤机体免疫抑制状态，疏肝健脾方具有治疗作用，中西医结合最为显著。基于细胞焦亡通路探索疏肝健脾方单纯或联合化疗治疗抑郁障碍型乳腺癌的作用机制，在于促进乳腺癌肿瘤组织内细胞焦亡，能够通过下调细胞焦亡相关细胞因子IL-1β、IL-18表达，调控细胞焦亡相关蛋白NLRP3、ASC和Caspase-1的表达水平，并下调TLR4/MyD88/NF-κB、Tim-3/Galectin-9/HMGB1等炎症通路蛋白表达水平，从而促进肿瘤细胞焦亡，抑制炎性微环境，达到防治乳腺癌发生发展及复发转移的目的；体外实验通过细胞共培养体系，证明MDSC细胞可促进4T1细胞增殖、4T1细胞抑制CD8+T细胞增殖，疏肝健脾方含药血清可通过细胞焦亡相关通路抑制4T1细胞增殖、抑制MDSC细胞功能，改善免疫抑制。.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。