剖宫产通过调节serpinB9甲基化介导肠粘膜Th17/Treg免疫失衡导致子代肠道炎症发生的机制研究

An increasing number of evidences have shown that cesarean section (CS) is associated with increased risk of inflammatory disease in the offspring, such as asthma, gastroenteritis, and inflammatory bowel disease, and the mechanisms of intestinal inflammation are unclear. The balance of Th17/Treg plays an important role in inflammatory diseases, and altered methylation has been reported to be involved in the development of inflammation. In our preliminary study, we found that methylation level of serpinB9 in cord blood of fetus delivered by CS was significantly decreased compared with vagina childbirth. RT-qPCR results indicated that the mRNA expression of serpinB9 was significantly upregulated in cord blood leukocytes of cesarean-born infants. Furthermore, RNA-seq analyses were performed to explore the transcriptomes and the expression profiles of different delivery modes, and IL-1β and MIF were found to have significantly increased in newborns by CS. All the current results in our lab suggest the up-regulation of serpinB9 promotes inflammation via upsetting the Th17/Treg balance. The objects of this project is first to analyze the DNA methylation level and mRNA expression level of serpinB9 in intestinal mucosa of young mouse born by CS. The second part will focus on investigating the up-regulation of serpinB9 will promote inflammation via upsetting the Th17/Treg balance, based on serpinB9 gene knock-in model. Thirdly, we will study the signaling pathways provoked by serpinB9, which induced the imbalance of Th17/Treg in intestinal mucosa by using protein interaction analysis, based on Casp1-/- and Il1b-/- gene knockout mice. In summary, the project’s aim is to reveal the mechanisms involving in the reduced methylation level of serpinB9 by CS leading to Th17/Treg imbalance and further intestinal inflammation, which will lay the foundation for developing a novel strategy of inflammatory disease therapy in the offspring.

剖宫产显著增加子代肠道炎症疾病风险，其作用机制不明。Th17/Treg平衡在炎症疾病中很重要，且甲基化异常与炎症发生密切相关。我们前期发现剖宫产可降低丝氨酸蛋白酶抑制因子serpinB9甲基化从而促进其表达；转录组发现介导Th17通路激活相关基因在剖宫产胎儿中高表达。据此我们推测剖宫产调节serpinB9甲基化介导Th17/Treg失衡引发子代肠道炎症。为验证该假说，本项目拟开展：1.利用剖宫产小鼠模型，明确剖宫产对仔鼠肠粘膜serpinB9甲基化和表达的影响；2.通过基因敲入小鼠，探讨serpinB9高表达介导Th17/Treg失衡促进仔鼠肠道炎症发生；3.采用基因敲除小鼠模型，阐明serpinB9介导Th17/Treg失衡的作用通路。以期阐明剖宫产通过调节子代serpinB9甲基化介导肠粘膜免疫失衡导致肠道炎症发生的分子机制，为以serpinB9为靶点治疗子代肠道炎症疾病提供理论依据。

我国是剖宫产率最高的国家之一，并且炎症性肠病（IBD）发病率迅速增高。剖宫产显著增加子代患炎症性疾病的风险，包括食物过敏、哮喘及炎症性肠病等，提示高剖宫产率可能增加子代IBD的发病风险。人群流行病调查研究发现，选择性剖宫产出生小孩儿童期患IBD风险显著高于阴道分娩出生小孩；然而剖宫产导致子代IBD易感性增加的机制还不清楚。丝氨酸蛋白酶抑制因子B9（SERPINB9）参与免疫、炎症发生，而我们前期研究发现SERPINB9的甲基化水平在剖宫产组显著降低，提示SERPINB9可能参与剖宫产介导的炎症反应。本项目通过构建剖宫产小鼠模型，证实剖宫产通过调控DNA甲基化水平介导仔鼠肠组织Serpinb9高表达；剖宫产通过影响仔鼠早期肠上皮细胞的分化、肠通透性改变及肠粘膜组织免疫细胞平衡异常，显著增加DSS诱导的结肠炎；单细胞转录组学发现SERPINB9在IBD患者肠粘膜组织高表达，主要分布在炎症性巨噬细胞；过表达SERPINB9，引起肠上皮细胞紧密连接蛋白ZO-1表达下调、细胞膜破裂及炎症因子IL-6释放增加；而敲除Serpinb9能通过激活Caspase1/GZMB-STAT3通路，改善小鼠肠上皮屏障损伤和粘膜免疫平衡，缓解小鼠结肠炎症；通过以上研究进一步从实验角度证实剖宫产增加IBD患病风险，并找到连接剖宫产和子代肠炎风险增加潜在分子机制，相关的研究成果有助于揭示剖宫产儿童肠道炎症疾病的发病机制。