基于单细胞转录组测序探讨仙连解毒方对结直肠癌湿热瘀毒证模型小鼠肠道免疫细胞的影响

Effect of Xianlian Jiedu Formula(仙连解毒方)on Intestinal Immune Cells in Colorectal Cancer Mice with Dampness-heat Stasis Toxin Syndrome based on Single-cell Transcriptome Sequencing

目的探讨仙连解毒方治疗结直肠癌湿热瘀毒证的可能作用机制。方法30只C57BL/6N小鼠随机分为正常组、模型组、仙连解毒方组,每组10只。模型组和仙连解毒方组小鼠采用高温高湿环境、高脂饮食与氧化偶氮甲烷/葡聚糖硫酸钠联合诱导建立结直肠癌湿热瘀毒证小鼠模型。造模成功后,仙连解毒方组给予仙连解毒方12.9 g/(kg·d)灌胃,正常组、模型组给予0.2 ml生理盐水灌胃,均每周6次,连续16周。给药期间每周统计疾病活动指数(DAI)评分,末次给药后处死小鼠,检测小鼠血清二胺氧化酶(DAO)、结直肠组织髓过氧化物酶(MPO)含量,取结肠组织进行单细胞转录组测序(scRNA-seq),采用SingleRv1.4.1,根据细胞表面标记基因,对肿瘤微环境(TME)中的免疫细胞进行细胞亚型鉴定,分析各组小鼠免疫细胞表达的差异基因的生物学功能和信号通路。结果仙连解毒方组给药第7周至第16周,DAI评分较模型组显著降低(P<0.01)。与正常组比较,模型组小鼠生存率显著降低,血清DAO、结直肠组织MPO水平显著升高(P<0.05或P<0.01);与模型组比较,仙连解毒方组小鼠生存率升高,血清DAO、结直肠组织MPO水平显著降低(P<0.05或P<0.01)。scRNA-seq分析结果显示,小鼠肠道T细胞可分为CD4^(+)T细胞、CD8^(+)T细胞、自然杀伤(NK)T细胞、γδT细胞4种亚型。与模型组比较,仙连解毒方组小鼠肠道组织中肠上皮细胞、成纤维细胞、红系细胞数量和占比升高,B细胞、T细胞、髓系细胞数量和占比降低;T细胞中激活CD4^(+)T细胞,效应CD4^(+)T细胞、记忆CD8^(+)T细胞、CD4^(+)T细胞、γδT细胞、NK T细胞数量和占比降低,细胞毒性CD8^(+)T细胞、耗竭CD8^(+)T细胞数量和占比升高,CD8^(+)T细胞、初始CD4^(+)T细胞、记忆CD4^(+)T细胞、调节性T细胞(Treg)、初始CD8^(+)T细胞数量降低,占比升高;髓系细胞中各类细胞数量皆下降,其中巨噬细胞与中性粒细胞占比降低,肥大细胞、树突状细胞占比升高。差异基因富集分析显示,仙连解毒方对CD8^(+)T细胞的调控功能主要富集在RNA聚合酶Ⅱ对转录的正向调控、转录因子激活蛋白-1(AP-1)复合物等,信号通路主要富集在破骨细胞分化、白细胞介素(IL-17)信号通路、核因子κB(NF-κB)信号通路、肿瘤坏死因子(TNF)信号通路等方面;对CD4^(+)T细胞的调控功能主要富集在伴侣辅因子依赖性蛋白质重折叠、p38丝裂原活化蛋白激酶(p38MAPK)级联负调控等,信号通路主要富集在炎症性肠病、破骨细胞分化、细胞凋亡、TNF信号通路等方面;对髓系细胞的调控功能主要富集在炎症反应、中性粒细胞趋化性、免疫反应等,信号通路主要富集在IL-17信号通路、TNF信号通路、NF-κB信号通路、抗原提呈加工等方面。结论仙连解毒方可能通过调控TME中的CD8^(+)T细胞、CD4^(+)T细胞、γδT细胞、巨噬细胞、中性粒细胞等多种免疫细胞数量和比例,干预Th1/Th2细胞分化、Th17细胞分化、IL-17、TNF、NF-κB等信号通路,改善结直肠癌免疫、炎性微环境,从而治疗结直肠癌湿热瘀毒证。

Objective To explore the possible mechanism of Xianlian Jiedu Formula(仙连解毒方,XJF)in treating dampness-heat stasis toxin syndrome of colorectal cancer.Methods Thirty C57BL/6N mice were randomly divided into normal group,model group and XJF group with 10 rats in each group.The mice in model group and XJF group were induced by high temperature and high humidity environment,high fat diet and azoxymethane/dextran sodi⁃um sulfate to establish the model of dampness-heat stasis toxin syndrome of colorectal cancer.After successful model⁃ing,XJF group was given 12.9 g/(kg·d)of XJF by gavage,while the normal group and the model group were given 0.2 ml of normal saline by gavage,both for 6 times per week,16 weeks.Disease activity index(DAI)score was cal⁃culated weekly during administration,and serum diamine oxidase(DAO)and colorectal tissue myeloperoxidase(MPO)contents were detected after last administration.The colonic tissue was taken for single-cell transcriptome se⁃quencing(scRNA-seq).According to the cell surface marker gene,SingleR-v1.4.1 was used to identify cell sub⁃types of the immune cells in the tumor microenvironment(TME).The biological function and signaling pathway of the differentially expressed genes by immune cells in each group were analyzed.Results Compared to that in the model group,DAI score in XJF group was significantly lower from week 7 to week 16 during administration(P<0.01).Compared to those in the normal group,the survival rate of mice in the model group significantly decreased,while the serum DAO and colorectal tissue MPO contents increased(P<0.05 or P<0.01);compared to the model group,the XJF group had higher survival rate and lower serum DAO and colorectal tissue MPO contents(P<0.05 or P<0.01).The scRNA-seq analysis showed that mouse intestinal T cells could be divided into four subtypes,including CD4^(+)T cells,CD8^(+)T cells,natural killer(NK)T cells andγδT cells.Compared to those in the model group,the number and proportion of intestinal epithelial cells,fibroblasts and erythroid cells in the intestinal tissues in XJF group increased,while the number and proportion of B cells,T cells and myeloid cells decreased;among T cells,the number and proportion of active CD4^(+)T cells,effector CD4^(+)T cells,memory CD8^(+)T cells,CD4^(+)T cells,γδT cells and NK T cells decreased,while those of CD8^(+)T cells,cytotoxic CD8^(+)T cells and depletion CD8^(+)T cells increased;the number of initial CD4^(+)T cells,memory CD4^(+)T cells,regulatory T cells(Tregs)and initial CD8^(+)T cells de⁃creased,while the proportion increased;the number of all kinds of myeloid cells decreased,while the proportion of macrophages and neutrophils decreased,and that of mast cells and dendritic cells increased.Differential gene enrich⁃ment analysis showed that the regulation function of XJF on CD8^(+)T cells was mainly concentrated in the positive regu⁃lation of RNA polymeraseⅡon transcription and transcription factor activator protein-1(AP-1)complex,and the sig⁃naling pathway was mainly concentrated in osteoclast differentiation,interleukin-17(IL-17)signaling pathway,nu⁃clear factorκB(NF-kB)signaling pathway,tumor necrosis factor(TNF)signaling pathway.The regulatory functions of CD4^(+)T cells was mainly concentrated in chaperone cofactory-dependent protein refolding,p38 mitogen-activated protein kinase(p38MAPK)cascade negative regulation,and the signaling pathways were mainly concentrated in in⁃flammatory bowel disease(IBD),osteoclast differentiation,cell apoptosis,and TNF signaling pathway.The regulatory functions of myeloid cells were mainly concentrated in inflammatory response,neutrophil chemotaxis,and immune re⁃sponse,and the signaling pathways were mainly concentrated in IL-17 signaling pathway,TNF signaling pathway,NF-kappa B signaling pathway,and antigen presentation and processing.Conclusion XJF can be used to treat colorectal cancer with dampness-heat stasis toxin syndrome,and its mechanism may be related to regulating the num⁃ber and proportion of CD8^(+)T cells,CD4^(+)T cells,γδT cells,macrophages,neutrophils and other immune cells in TME,and interfering with Th1/Th2 cell differentiation,Th17 cell differentiation,IL-17,TNF,NF-κB and other sig⁃naling pathways,so as to improve the immune and inflammatory microenvironment of colorectal cancer.