肠道微生物代谢产物上调lncRNA在肾脏衰老中的作用机制研究

It is known that the age-related perturbation in the gut microbiome can lead to the excessive production of microbial metabolites indoxyl sulfate (IS) and p-cresol sulfate (PCS), and the increased IS and PCS subsequently induce the inflammatory reaction. The results of our preliminary experiment demonstrate IS and PCS can up-regulate long noncoding RNA (IncRNA) Arid2-IR and NF-κB/p65 expression in HK-2 cell. Therefore, we speculate that IS and PCS can increase the production of senescence-associated secretory phenotype (SASP) to accelerates the renal aging by activating lncRNA Arid2-IR-NF-κB signaling pathway. In this study, first of all, we will verify whether IS and PCS can up-regulate the expression of lncRNA Arid2-IR to promote renal inflammation and aging; Further, via silencing and overexpressing lncRNA Arid2-IR, as well as knocking out and knocking in NF-κB target gene in cells and animal models, we will elucidate the mechanisms by which lncRNA Arid2-IR activates the NF-κB-SASP signaling pathway to promote renal aging; Then, Fecal microbiota transplantation and clinical research are applied to clarify the role of the above signaling pathway in renal aging again, and also the clinical value of removing IS and PCS in delaying renal aging. Generally, this study will give an new insight into elucidating the pathogenesis of renal aging, and thus provide the theoretical foundation and clinical guidance for preventing renal aging and attenuating inflammatory injury.

衰老时肠道菌群紊乱致其代谢产物硫酸吲哚酚（IS）、硫酸对甲酚（PCS）增多介导肾脏炎症反应，前期研究发现IS、PCS刺激HK-2细胞lncRNA Arid2-IR和NF-κB/p65表达升高，推测IS、PCS通过上调lncRNA Arid2-IR活化NF-κB信号通路致使衰老相关分泌表型（SASP）增多加速肾脏衰老。本项目首先从体内外实验验证IS、PCS上调lncRNA Arid2-IR促进肾脏炎症及衰老的作用；继而在特异性lncRNA Arid2-IR、NF-κB靶基因敲除及过表达细胞和动物模型上解析lncRNA Arid2-IR调节NF-κB升高SASP的作用机制；最后通过小鼠粪菌移植和临床验证阐明上述信号通路在肾脏衰老中的地位及减少IS、PCS产生对延缓肾脏衰老的临床价值。结果不但丰富人们对肾脏衰老发病机制的认识，也从改善肠道微生态角度为预防及延缓肾脏衰老和降低炎症反应提供新的靶点。

肾脏衰老可促进急慢性肾脏病的进展，但目前对于肾脏衰老的分子机制研究尚不明确。非编码RNA，包括环状RNA（circRNA）和长链非编码RNA（lncRNA）在肾脏疾病及器官衰老中发挥了重要的作用，尚无研究报道非编码RNA在肾脏衰老中的作用。肠道微生物代谢产物氧化三甲胺（TMAO）在机体衰老时产生增加，可促进肾间质纤维化的进展，也可诱导器官衰老的发生，提示其可能在肾脏衰老进程中发挥重要作用。TMAO也可通过调控长链非编码RNA的表达参与疾病的进展过程，但TMAO能否通过调控非编码RNA，进而影响肾脏衰老仍不清楚。因此，本项目一共分为两部分展开研究。第一部分研究通过全转录组测序、生信分析和分子生物学手段筛选并且验证了不同月龄小鼠肾脏中差异表达的circRNA，随后构建了差异circRNA相关的ceRNA网络，筛选出该网络中的核心调控分子circVmn2r1，它可能在肾脏衰老中扮演重要角色。随后通过细胞及动物实验验证了circVmn2r1能通过ceRNA机制，海绵结合miR-223-3p，上调NLRP3蛋白的表达，促进TCMK-1细胞衰老的发生。最后通过体外细胞实验发现TMAO能促进TCMK-1细胞衰老并且上调circVmn2r1的表达，敲低circVmn2r1可部分逆转TMAO诱导的细胞衰老作用，从而得出TMAO能通过上调circVmn2r1的表达促进TCMK-1细胞衰老的机制。第二部分研究通过全转录组测序和生物信息学分析方法对不同月龄小鼠肾脏组织中差异表达的lncRNA和mRNA进行分析，筛选随小鼠月龄增长差异表达的lncRNA，并对其靶基因进行GO和KEGG分析，明确3月龄和24月龄相比差异更加明显，进而对3月龄和24月龄小鼠肾脏组织中差异表达mRNA进行分析，然后构建ceRNA网络和lncRNA-mRNA互作网络，选择网络核心分子lncRNA Gm44981进行接下来的研究。结果发现其主要定位于小鼠肾小球系膜细胞的细胞核中，通过作用于P21加速细胞周期进展和促进细胞增殖；通过体内外实验阐明lncRNA Gm44981通过招募EZH2至P21的启动子区导致启动子区甲基化从而抑制P21转录，进而抑制肾小球系膜细胞衰老的分子机制。本研究将肠道微生物代谢产物和非编码RNA联系起来，为他们参与肾脏衰老的发病机制提供了科学依据，从而为延缓肾脏衰老的进展提供了新的治疗靶点。