骨髓间充质干细胞衍生的外泌体 miR-30e-5p 通过抑制 ELAVL1 改善高糖诱导的肾近端小管细胞焦亡。

BACKGROUND: The rapid increase in the prevalence of diabetes has resulted in more cases of diabetic kidney disease (DKD). Treatment with bone marrow mesenchymal stem cells (BMSCs) may represent an alternative strategy to manage DKD. METHODS: HK-2 cells were treated with 30 mM high glucose (HG). Bone marrow MSC-derived exosomes (BMSC-exos) were isolated and internalized into HK-2 cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) and lactate dehydrogenase (LDH) assays were used to measure viability and cytotoxicity. The secretion of IL-1β and IL-18 was measured by ELISA. Pyroptosis was assessed by flow cytometry. Quantitative RT-PCR was used to measure the levels of miR-30e-5p, ELAV like RNA binding protein 1 (ELAVL1), IL-1β, and IL-18. The expression of ELAVL1 and pyroptosis-associated cytokine proteins was determined by western blot analysis. A dual-luciferase reporter gene assay was conducted to confirm the relationship between miR-30e-5p and ELAVL1. RESULTS: BMSC-exos decreased LDH, IL-1β, and IL-18 secretion and inhibited the expression of the pyroptosis-related factors (IL-1β, caspase-1, GSDMD-N, and NLRP3) in HG-induced HK-2 cells. Moreover, miR-30e-5p depletion derived from BMSC-exos promoted HK-2 cell pyroptosis. Besides, miR-30e-5p over-expression or ELVAL1 knockdown could directly inhibit pyroptosis. ELAVL1 was a target of miR-30e-5p and knocking down ELAVL1 reversed the effect of miR-30e-5p inhibition in BMSC-exos-treated HK-2 cells. CONCLUSIONS: BMSC-derived exosomal miR-30e-5p inhibits caspase-1-mediated pyroptosis by targeting ELAVL1 in HG-induced HK-2 cells, which might provide a new strategy for treating DKD.

背景：糖尿病患病率的迅速上升导致了更多的糖尿病肾病（DKD）病例。骨髓间充质干细胞（BMSCs）的治疗可能代表了一种管理 DKD 的替代策略。

方法：用 30mmol/L 高糖（HG）处理 HK-2 细胞。分离骨髓间充质干细胞来源的外泌体（BMSC-exos）并将其内化到 HK-2 细胞中。用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）和乳酸脱氢酶（LDH）测定法测量细胞活力和细胞毒性。通过酶联免疫吸附试验（ELISA）测量 IL-1β 和 IL-18 的分泌。通过流式细胞术评估细胞焦亡。用定量 RT-PCR 测量 miR-30e-5p、ELAV 样 RNA 结合蛋白 1（ELAVL1）、IL-1β 和 IL-18 的水平。通过 Western blot 分析测定 ELAVL1 和细胞焦亡相关细胞因子蛋白的表达。通过双荧光素酶报告基因实验证实 miR-30e-5p 与 ELAVL1 之间的关系。

结果：BMSC-exos 降低了 HG 诱导的 HK-2 细胞中 LDH、IL-1β 和 IL-18 的分泌，并抑制了与细胞焦亡相关的因子（IL-1β、caspase-1、GSDMD-N 和 NLRP3）的表达。此外，BMSC-exos 衍生的 miR-30e-5p 耗竭促进了 HK-2 细胞的细胞焦亡。此外，miR-30e-5p 的过表达或 ELVAL1 的敲低可以直接抑制细胞焦亡。ELAVL1 是 miR-30e-5p 的靶标，敲低 ELAVL1 逆转了 BMSC-exos 处理的 HK-2 细胞中 miR-30e-5p 抑制的作用。

结论：BMSC 衍生的外泌体 miR-30e-5p 通过靶向 HG 诱导的 HK-2 细胞中的 ELAVL1 抑制 caspase-1 介导的细胞焦亡，这可能为治疗 DKD 提供一种新的策略。