人脐带间充质干细胞外泌体通过上调ATF3抑制TLR4/NF-κB通路减轻肾缺血-再灌注损伤

Human umbilical cord mesenchymal stem cell-derived exosomes attenuate renal ischemia-reperfusion injury by up-regulating ATF3 to inhibit the TLR4/NF-κB pathway

    摘要
  • 摘要:
    目的  探讨人脐带间充质干细胞来源外泌体（hucMSC-Exo）对肾缺血-再灌注损伤（IRI）的保护作用及机制。
    方法 分离并鉴定hucMSC-Exo。建立小鼠肾IRI模型，分为假手术组（Sham组）、IRI组、IRI+hucMSC-Exo组、IRI+hucMSC-Exo+JY-2组、Sham+JY-2组。检测各组小鼠血清肌酐（Scr）和血尿素氮（BUN）水平，苏木素-伊红（HE）染色观察肾组织病理变化，酶联免疫吸附试验检测各组小鼠血清白细胞介素（IL）-1β和IL-18水平，蛋白质印迹法检测激活转录因子3（ATF3）、Toll样受体4（TLR4）、核因子（NF）-κB、NOD样受体蛋白3（NLRP3）、半胱氨酸天冬氨酸蛋白酶1（Caspase-1）、Gasdermin D（GSDMD）的表达，实时荧光定量聚合酶链反应检测ATF3、TLR4、NF-κB信使RNA（mRNA）表达，免疫组织化学检测NLRP3、Caspase-1 p20、GSDMD表达。建立HK-2细胞缺氧/复氧（H/R）模型，分为对照组（Control组）、H/R组、H/R+hucMSC-Exo组、H/R+hucMSC-Exo+JY-2组、Control+JY-2组。蛋白质印迹法检测各组ATF3、TLR4、NF-κB蛋白表达水平，实时荧光定量聚合酶链反应检测NLRP3、GSDMD、Caspase-1 mRNA表达水平。
    结果 成功分离并鉴定出hucMSC-Exo。与Sham组比较，IRI组小鼠血清Scr和BUN水平升高，肾小管损伤评分升高，ATF3、TLR4、NF-κB p65、NLRP3、Caspase-1 p20、GSDMD蛋白表达水平升高，ATF3、TLR4、NF-κB mRNA表达水平升高；与IRI组比较，IRI+hucMSC-Exo组血清Scr和BUN水平下降，肾小管损伤评分下降，ATF3蛋白表达水平升高，TLR4、NF-κB p65、NLRP3、Caspase-1 p20、GSDMD蛋白表达水平下降，ATF3 mRNA表达水平升高，TLR4、NF-κB mRNA表达水平下降；与IRI+hucMSC-Exo组比较，IRI+hucMSC-Exo+JY-2组Scr和BUN水平升高，肾小管损伤评分升高，ATF3蛋白表达水平下降，TLR4、NF-κB p65、NLRP3、Caspase-1 p20、GSDMD蛋白表达水平升高，ATF3 mRNA表达水平下降，TLR4、NF-κB mRNA表达水平升高（均为P<0.05）。与Control组比较，H/R组ATF3、TLR4及NF-κB p65蛋白表达水平升高，NLRP3、Caspase-1 和GSDMD mRNA表达水平升高；与H/R组比较，H/R+hucMSC-Exo组ATF3蛋白表达水平升高，TLR4和NF-κB p65蛋白表达水平下降，NLRP3、Caspase-1 和GSDMD mRNA表达水平下降；与H/R+hucMSC-Exo组比较，H/R+hucMSC-Exo+JY-2组ATF3蛋白表达水平下降，TLR4和NF-κB p65蛋白表达水平升高，NLRP3、Caspase-1 和GSDMD mRNA表达水平升高（均为P<0.05）。
    结论 HucMSC-Exo可通过上调ATF3表达，负向调控TLR4/NF-κB信号通路，进而抑制细胞焦亡，最终减轻肾IRI。

     

    Abstract:
    Objective To investigate the protective effect and underlying mechanism of human umbilical cord mesenchymal stem cell-derived exosomes (hucMSC-Exo) on renal ischemia-reperfusion injury (IRI).
    Methods hucMSC-Exos were isolated and characterized. A mouse renal IRI model was established and the animals were divided into Sham, IRI, IRI+hucMSC-Exo, IRI+hucMSC-Exo+JY-2 and Sham+JY-2 groups. Serum creatinine (Scr) and blood urea nitrogen (BUN) were measured. Hematoxylin-eosin (HE) staining was used to evaluate renal histopathology. Enzyme-linked immune absorbent assay was performed to determine serum interleukin (IL)-1β and IL-18 levels. Western blotting was used to detect the expression of activating transcription factor 3 (ATF3), Toll-like receptor 4 (TLR4), nuclear factor (NF)-κB, NOD-like receptor protein 3 (NLRP3), cysteineyl aspartate specific proteinase (Caspase)-1 p20 and Gasdermin D(GSDMD). Real-time fluorescent quantitative polymerase chain reaction was employed to measure ATF3, TLR4 and NF-κB messenger RNA (mRNA). Immunohistochemistry was conducted to examine NLRP3, Caspase-1 p20 and GSDMD. An in vitro hypoxia/reoxygenation (H/R) model was established in HK-2 cells and divided into Control, H/R, H/R+hucMSC-Exo, H/R+hucMSC-Exo+JY-2 and Control+JY-2 groups. Western blotting was used to detect the expression of ATF3, TLR4 and NF-κB. Real-time fluorescent quantitative polymerase chain reaction was used to measure NLRP3, GSDMD and Caspase-1 mRNA.
    Results HucMSC-Exos were successfully isolated and identified. Compared with the Sham group, the IRI group exhibited elevated Scr and BUN, higher tubular injury scores, increased protein expression levels of ATF3, TLR4, NF-κB p65, NLRP3, Caspase-1 p20 and GSDMD, and raised mRNA expression levels of ATF3, TLR4, NF-κB. Compared with the IRI group, the IRI+hucMSC-Exo group showed decreased Scr and BUN, lower tubular injury scores, up-regulated ATF3 protein and mRNA, down-regulated TLR4, NF-κB p65, NLRP3, Caspase-1 p20 and GSDMD protein, and declined TLR4 and NF-κB mRNA. Compared with the IRI+hucMSC-Exo group, the IRI+hucMSC-Exo+JY-2 group exhibited increased Scr and BUN levels, elevated renal tubular injury scores, decreased ATF3 protein expression levels, elevated protein expression levels of TLR4, NF-κB p65, NLRP3, Caspase-1 p20, and GSDMD, decreased ATF3 mRNA expression levels, and elevated mRNA expression levels of TLR4 and NF-κB. (all P < 0.05). Compared with the Control group, the expression levels of ATF3, TLR4 and NF-κB p65 proteins were increased in the H/R group, and the expression levels of NLRP3, Caspase-1 and GSDMD mRNA were increased. Compared with the H/R group, the expression level of ATF3 protein was increased, the expression levels of TLR4 and NF-κB p65 proteins were decreased, and the expression levels of NLRP3, Caspase-1 and GSDMD mRNA were decreased in the H/R+hucMSC-Exo group. Compared with the H/R+hucMSC-Exo group, the expression level of ATF3 protein was decreased, the expression levels of TLR4 and NF-κB p65 proteins were increased, and the expression levels of NLRP3, Caspase-1 and GSDMD mRNA were increased in the H/R+hucMSC-Exo+JY-2 group (all P < 0.05).
    Conclusions  HucMSC-Exos alleviate renal IRI by up-regulating ATF3, thereby negatively regulating the TLR4/NF-κB signaling pathway and subsequently inhibiting pyroptosis.

     

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