巨噬细胞来源外泌体miR-330-3p介导糖尿病足细胞焦亡的机制研究

Pyroptosis is inflammation-mediated programmed cell death, and it has been confirmed that podocytes pyroptosis is closely related to diabetic kidney disease. Thioredoxin-interacting protein(TXNIP) is tightly associated with various biological and pathological processes, including oxidative stress, inflammation, fibrosis, as well as cell pyroptosis. Our pre-study suggested that forkhead transcription factor O1 (FoxO1) could restrain pyroptosis by inhibiting the expression of TXNIP in podocytes; however, the exact mechanism involved has not been elucidated yet. Thus, we have accomplished the microRNA microarray analysis of plasma exosomes from patients with or without diabetic kidney disease, followed by proceeding bioinformatics analysis, literature review and functional experiments. The results suggested that the increased level of miR-330-3p in plasma exosomes might mediate podocytes pyroptosis, probably by targeting FoxO1. In consideration of macrophages which were closely related to renal inflammation, and miR-330-3p was upregulated in macrophages under high glucose by literature review, we further extracted the exosomes from high glucose cultured-macrophages and found that the level of miR-330-3p was significantly increased in both high glucose cultured-macrophages and exosomes in cultured-supernate. After co-cultured with macrophage exosomes, mir-330-3p in podocytes was significantly increased, accompanied by decreased FoxO1 expression and elevated caspase-1 level. Hence, we hypothesized that miR-330-3p in exosomes from high glucose-induced macrophages might mediate podocyte pyroptosis by regulating FoxO1/TXNIP pathway. Therefore, podocyte-based assays and molecular approaches will be used to explore the impacts and mechanisms of miR-330-3p in exosomes from high glucose cultured-macrophages on podocyte pyroptosis, including CRISPR/Cas9, exosome fluorescent tracing, co-immunoprecipitation and dual luciferase reporter gene assay, etc.; following by utilizing exosomes renal cortex injection, lipsomal clodronate injection, in vivo bioluminescence imaging and other techniques on C57/BL6 mice or kidney-specific FoxO1 transgenic mice, so as to validate the effects of macrophage-derived exosomes on podocyte pyroptosis in diabetic mice. This project will shed light on novel strategy of preventing and controlling diabetic kidney disease.

焦亡是炎症反应介导的细胞程序性死亡方式，已证实足细胞焦亡与糖尿病肾病密切相关；FoxO1可抑制TXNIP表达而减少足细胞焦亡，但上游机制并不明确。我们前期分析糖尿病肾病患者血外泌体miRNA芯片表达谱，发现显著增高的miR-330-3p可能介导足细胞焦亡，且FoxO1为预测靶基因；进一步检测巨噬细胞培养上清液外泌体，发现其中miR-330-3p水平明显升高，以此外泌体干预足细胞可致miR-330-3p升高、FoxO1下降。因此我们假设：巨噬细胞来源外泌体中miR-330-3p可能通过调节FoxO1/TXNIP通路介导足细胞焦亡发生。为此，本项目拟提取巨噬细胞来源外泌体，应用CRISPR/Cas9、外泌体荧光示踪、免疫共沉淀及荧光素酶报告基因等技术，揭示巨噬细胞来源外泌体中miR-330-3p介导足细胞焦亡的具体机制，并借助小动物活体成像等技术于小鼠体内加以验证，为糖尿病肾病防治提供新策略。

焦亡是炎症反应介导的细胞程序性死亡方式，已证实足细胞焦亡与糖尿病肾病（DKD）密切相关。前期研究发现，高糖培养的巨噬细胞来源外泌体中miR-21-5p的表达显著增高，以此外泌体干预足细胞可致miR-21-5p升高，足细胞焦亡相关蛋白及炎症因子表达升高，足细胞损伤加重。A20作为抗炎因子，也是生信软件预测到的miR-21-5p潜在结合位点，很可能参与了这一过程。但高糖培养的巨噬细胞来源外泌体诱导足细胞损伤的具体机制尚不清楚。本课题使用miR-21-5p mimic、miR-21-5p inhibitor、si-A20转染细胞，应用荧光素酶报告基因等技术，在细胞和动物水平上揭示高糖培养的巨噬细胞来源外泌体的miR-21-5p通过靶向A20调节炎症途径介导的足细胞损伤，为寻找新的DKD治疗靶点提供新思路。早期研究发现，尿外泌体miRNA比血外泌体miRNA更有资格作为DKD早期诊断和治疗的非侵入性生物标志物。本课题通过对2型糖尿病长病程无肾病患者以及及具有相似病程合并糖尿病肾病患者尿外泌体miRNA芯片表达谱测定并结合生物信息学分析，筛选出miR-4534作为早期DKD的生物学标志,为DKD的早期诊断提供新思路，以便在疾病初期干预治疗改善预后效果。临床诊治发现，2型糖尿病患者并发慢性肾脏疾病(CKD)不仅可以是DKD，也存在非糖尿病性肾脏疾病(NDRD)或DKD和NDRD混合(MIX)的可能。本课题通过开展前瞻性观察性研究探讨肾活检证实的DKD、NDRD和MIX的临床特征和预后差异，并确定不良肾脏结局的基线危险因素。发现NDRD组患者的肾脏预后优于单纯DKD组和MIX组；肾功能恶化、严重蛋白尿、血红蛋白降低和有糖尿病家族史可能与DKD患者的不良肾脏结局有关。这些指标的评估有助于识别可能具有不良肾脏预后的患者，为DKD早期预警提供支持。