微小RNA-150-5p通过抑制丝裂原活化蛋白激酶激酶激酶3/应激活化蛋白激酶途径来预防脓毒症急性肾损伤。

MiR-150-5p protects against septic acute kidney injury via repressing the MEKK3/JNK pathway.

作者信息

Shi Lang, Zhang Yafei, Xia Yao, Li Chenglong, Song Zhixia, Zhu Jiefu

机构信息

Department of Nephrology, The First Clinical Medical College of Three Gorges University, Center People's Hospital of Yichang, Yichang, Hubei 443000, China.

Department of Urology, Renmin Hospital of Wuhan University, Wuhan, Hubei 430060, China.

出版信息

Cell Signal. 2021 Oct;86:110101. doi: 10.1016/j.cellsig.2021.110101. Epub 2021 Jul 30.

DOI:10.1016/j.cellsig.2021.110101

PMID:34333083

Abstract

BACKGROUND

Septic acute kidney injury (AKI) is associated with increased morbidity and mortality in critically ill patients. MicroRNA is reportedly involved in sepsis-induced organ dysfunction, while the role of miR-150 in septic AKI remains ambiguous.

METHODS

Quantitative real-time PCR (qRT-PCR) was carried out to examine miR-150-5p expression in both septic AKI patients and volunteers without septic AKI. Lipopolysaccharide (LPS) was used to treat renal tubular epithelial cell line HK-2 and C57/BL6 mice to establish in vitro and in vivo sepsis-induced AKI models. Cell apoptosis was determined using TdT-mediated dUTP nick end labeling (TUNEL) staining and flow cytometry. Cell viability was tested using a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Renal pathological changes were examined via Hematoxylin-Eosin (H&E) staining, and renal function was measured via blood urea nitrogen (BUN) and creatinine (Cre) measurements. The MEKK3/JNK profile and oxidative stress markers (including COX2 and iNOS) were examined by immunoblot analysis, and the expression levels of inflammatory cytokines (TNF-α, IL-6, and IL-1β) and oxidative stress markers (MDA, SOD, and CAT) were evaluated by ELISA.

RESULTS

MiR-150-5p was down-regulated in the serum of patients with septic AKI (compared to healthy volunteers). Moreover, miR-150-5p levels were lower in LPS-treated HK-2 cell lines and in the septic AKI mouse model. Additionally, Stat-3 activation mediated the decrease of miR-150-5p. Functionally, miR-150-5p agomir attenuated LPS-induced apoptosis in HK-2 cells, in addition to renal inflammatory responses and oxidative stress. In contrast, inhibition of miR-150-5p aggravated LPS-induced apoptosis, inflammatory reactions and oxidative stress. Furthermore, miR-150-5p agomir decreased BUN and Scr levels in the septic AKI mice model repressed TNF-α, IL-6 and IL-1β, and up-regulated SOD and CAT down-regulated MDA in the kidney tissues. Moreover, miR-150-5p was identified as a target gene for Stat3, and the overexpression of Stat3 partially promoted the effect of down-regulating miR-150-5p on LPS-induced HK2 cell injury. Mechanistically, the MEKK3/JNK pathway was identified as a functional target of miR-150-5p, and the knockdown of MEKK3 showed protective effects against LPS mediated HK-2 cell apoptosis.

CONCLUSION

Stat3-mediated miR-150-5p exerted protective effects in sepsis-induced acute kidney injury by regulating the MEKK3/JNK pathway.

摘要

背景

脓毒症性急性肾损伤（AKI）与危重症患者发病率和死亡率的增加相关。据报道，微小RNA参与脓毒症诱导的器官功能障碍，而miR-150在脓毒症性AKI中的作用仍不明确。

方法

采用定量实时聚合酶链反应（qRT-PCR）检测脓毒症性AKI患者和无脓毒症性AKI志愿者血清中miR-150-5p的表达。用脂多糖（LPS）处理肾小管上皮细胞系HK-2和C57/BL6小鼠，建立体外和体内脓毒症诱导的AKI模型。采用TdT介导的dUTP缺口末端标记（TUNEL）染色和流式细胞术检测细胞凋亡。用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐（MTT）法检测细胞活力。通过苏木精-伊红（H&E）染色检查肾脏病理变化，并通过检测血尿素氮（BUN）和肌酐（Cre）来测定肾功能。通过免疫印迹分析检测MEKK3/JNK信号通路和氧化应激标志物（包括COX2和iNOS），并通过酶联免疫吸附测定（ELISA）评估炎性细胞因子（TNF-α、IL-6和IL-1β）和氧化应激标志物（MDA、SOD和CAT）的表达水平。

结果

脓毒症性AKI患者血清中miR-150-5p表达下调（与健康志愿者相比）。此外，LPS处理的HK-2细胞系和脓毒症性AKI小鼠模型中miR-150-5p水平较低。此外，Stat-3激活介导了miR-150-5p的降低。在功能上，miR-150-5p激动剂除了减轻肾脏炎症反应和氧化应激外，还减轻了LPS诱导的HK-2细胞凋亡。相反，抑制miR-150-5p会加重LPS诱导的细胞凋亡、炎症反应和氧化应激。此外，miR-150-5p激动剂降低了脓毒症性AKI小鼠模型中的BUN和Scr水平，抑制了TNF-α、IL-6和IL-1β，并上调了肾脏组织中的SOD和CAT，下调了MDA。此外，miR-150-5p被鉴定为Stat3的靶基因，Stat3的过表达部分促进了下调miR-150-5p对LPS诱导的HK2细胞损伤的影响。机制上，MEKK3/JNK信号通路被鉴定为miR-150-5p的功能靶点，敲低MEKK3对LPS介导的HK-2细胞凋亡具有保护作用。